WO2002044994A2 - Systemes et procedes de commande, de conception, de production, d'inventaire, de vente et d'analyse de dosages de detection, pouvant etre utilises avec ou dans un moyen de production - Google Patents

Systemes et procedes de commande, de conception, de production, d'inventaire, de vente et d'analyse de dosages de detection, pouvant etre utilises avec ou dans un moyen de production Download PDF

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Publication number
WO2002044994A2
WO2002044994A2 PCT/US2001/045705 US0145705W WO0244994A2 WO 2002044994 A2 WO2002044994 A2 WO 2002044994A2 US 0145705 W US0145705 W US 0145705W WO 0244994 A2 WO0244994 A2 WO 0244994A2
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WIPO (PCT)
Prior art keywords
assay
detection
data
synthesizer
component
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Application number
PCT/US2001/045705
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English (en)
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WO2002044994A3 (fr
WO2002044994A9 (fr
WO2002044994A8 (fr
Inventor
Amy Brower
Mary Ann Brow
Raymond F. Cracauer
Lance Fors
Rocky Granske
Monika De Arruda Indig
David Kurensky
Craig Luedtke
Andrew A. Lukowiak
Victor Lyamichev
Bruce P. Neri
Ned D. Reimer
Robert T. P. Roeven
Zbiginiev Skrzypczynski
Witold A. Ziarno
John Comerford
Steven Stump
Daniel D. Viegut
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Third Wave Technologies, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/771,332 external-priority patent/US6932943B1/en
Priority claimed from US09/915,063 external-priority patent/US20030082544A1/en
Priority claimed from US09/929,135 external-priority patent/US20030104470A1/en
Priority claimed from US09/930,535 external-priority patent/US20030072689A1/en
Priority claimed from US09/930,646 external-priority patent/US20030113237A1/en
Priority claimed from US09/930,543 external-priority patent/US20030113236A1/en
Priority claimed from US09/930,688 external-priority patent/US20030124526A1/en
Priority claimed from US10/002,251 external-priority patent/US20020156255A1/en
Priority to AU3945502A priority Critical patent/AU3945502A/xx
Priority claimed from US10/054,023 external-priority patent/US7435390B2/en
Priority to JP2002547086A priority patent/JP2004536562A/ja
Application filed by Third Wave Technologies, Inc. filed Critical Third Wave Technologies, Inc.
Priority to AU2002239455A priority patent/AU2002239455A1/en
Priority to EP01987217A priority patent/EP1364334A2/fr
Publication of WO2002044994A2 publication Critical patent/WO2002044994A2/fr
Publication of WO2002044994A8 publication Critical patent/WO2002044994A8/fr
Publication of WO2002044994A3 publication Critical patent/WO2002044994A3/fr
Publication of WO2002044994A9 publication Critical patent/WO2002044994A9/fr

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    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06Q—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES; SYSTEMS OR METHODS SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES, NOT OTHERWISE PROVIDED FOR
    • G06Q10/00—Administration; Management
    • G06Q10/08—Logistics, e.g. warehousing, loading or distribution; Inventory or stock management
    • G06Q10/087—Inventory or stock management, e.g. order filling, procurement or balancing against orders
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06Q—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES; SYSTEMS OR METHODS SPECIALLY ADAPTED FOR ADMINISTRATIVE, COMMERCIAL, FINANCIAL, MANAGERIAL OR SUPERVISORY PURPOSES, NOT OTHERWISE PROVIDED FOR
    • G06Q30/00—Commerce
    • G06Q30/06—Buying, selling or leasing transactions

Definitions

  • the present invention relates to detection assay ordering, development, production, sales and optimization methods and systems for the commercialization of products, such as research use only products (RUOs), analyte specific reagents (ASRs) and in vitro diagnostics (INDs).
  • products such as research use only products (RUOs), analyte specific reagents (ASRs) and in vitro diagnostics (INDs).
  • genomics research and subsequent drug design efforts increase as well.
  • a number of institutions are actively mining the available genetic sequence information to identify correlations between genes, gene expression and phenotypes (e.g., disease states, metabolic responses, and the like). These analyses include an attempt to characterize the effect of gene mutations and genetic and gene expression heterogeneity in individuals and populations.
  • information on the frequency and clinical relevance of many polymorphisms and other variations has yet to be obtained and validated.
  • the human reference sequences used in current genome sequencing efforts do not represent an exact match for any one person's genome.
  • HGP Human Genome Project
  • researchers collected blood (female) or sperm (male) samples f om a large number of donors.
  • sperm male samples
  • the human genome sequence generated by the private genomics company Celera was based on DNA samples collected from five donors who identified themselves as Hispanic, Asian, Caucasian, or African- American.
  • the small number of human samples used to generate the reference sequences does not reflect the genetic diversity among population groups and individuals. Attempts to analyze individuals based on the genome sequence information will often fail.
  • probe oligonucleotides For example, many genetic detection assays are based on the hybridization of probe oligonucleotides to a target region on genomic DNA or mRNA. Probes generated based on the reference sequences will often fail (e.g., fail to hybridize properly, fail to properly characterize the sequence at specific position of the target) because the target sequence for many individuals differs from the reference sequence. Differences may be on an individual-by-individual basis, but many follow regional population patterns (e.g., many correlate highly to race, ethnicity, geographic local, age, environmental exposure, etc.).
  • the invention provides systems and methods for ordering, manufacturing and selling detection assays, and instrumentation related thereto.
  • the system includes one or more components, such as a computer-based customer order component for ordering at least one of a plurality of oligonucleotide detection assays, and/or related instrumentation; a detection assay production component for creating the oligonucleotide detection assays; a shipping component for shipping said oligonucleotide detection assays and/or related instrumentation; and a billing component for billing a customer for the oligonucleotide detection assays and/or related instrumentation.
  • the billing component comprises a payment receipt component for receiving payment for the oligonucleotide detection assays.
  • the present invention further provides systems, methods, and compositions that provide comprehensive solutions for the manufacturing, use, analysis, and sales of detection assays (e.g., oligonucleotide detection assays).
  • detection assays e.g., oligonucleotide detection assays
  • the present invention provides systems and methods for the ordering of detection assay, including electronic ordering (e.g., over public or private electronic communication networks) by general customers, as well as, distributors, collaborators, health care professionals, individuals, and established long-term customers.
  • detection assay design including electronic quality assessment methods of detection assay components and design of primers (e.g., amplification primers) and probes.
  • Assay design is made possible for large numbers of diverse assays (of a single type or of multiple types) and for large-scale production thereof, including the design of panels, research products, and clinical products (e.g., in vitro diagnostic products).
  • the present invention also provides systems and methods for detection assay production, including coordinated synthesis, preparation, and quality control of detection assay components, and also detection assay assembly on a variety of presentation platforms, including 96, 384, 1536 well plates, and combinations thereof, slides, and other presentation platforms. Inventory control systems and methods, and design and production management systems and methods, are also provided for complete detection assays, for detection assay components, reagents for the creation of detection assays, and instrumentation used to manufacture detection assays.
  • the present invention also provides systems and methods for selling detection assays, and systems and methods for assisting detection assay users in the collection and analysis of data produced by the use of the detection assays (of a single variety or of multiple varieties).
  • the present invention also provides systems and methods for collecting, analyzing, and storing data, including detection assay design data and data generated by the use of the detection assays.
  • Each of the components of the systems and methods of the present invention may be integrated to provide comprehensive systems and methods for the manufacture and use of detection assays, with exchange of data between various components of the system to optimize utilization of the data generated by the detection assay or detection assay usage. Integration provides, by way of further example, methods to coordinate the movement of genetic information from research applications to in vitro diagnostic applications.
  • the computer based customer order entry component further comprises a consumer direct web order entry component. Consumers, include by way of example, the purchasing public.
  • the computer based customer order entry component further includes home or work computers, workstations, PDAs or web appliances of members of the public.
  • the computer-based customer order entry component provides a unidirectional, bi-directional or omni-directional data feed into the detection assay production component, other components of the system and/or portions thereof. In certain embodiments, the data feed affects production cycles of the oligonucleotide detection assays.
  • the data feed comprises statistical information associated with or related to one or more oligonucleotide detection assays of a single variety or one or more oligonucleotide detection assays of one or more varieties.
  • the statistical information is selected from the group consisting of total oligonucleotide detection assays ordered or oligonucleotide detection assay orders received; a histogram; an oligonucleotide detection assay average per consumer; an arithmetic mean; quantity of oligonucleotide detection assays, size of order of oligonucleotide detection assays; format of panel information; a mode; a median; a weighted mean; a harmonic mean; a geometric mean; a logarithmic mean; a root mean square; a root sum square, and combination thereof; a normal distribution curve, the normal distribution curve includes, but is not limited to, a normal distribution curve of number of consumers, number of detection assays, quantity
  • the present invention provides a system and method for manufacturing and selling detection assays, comprising one or more of the following components: a computer-based customer order component for ordering at least one of a plurality of oligonucleotide detection assays; a detection assay production component for creating the ohgonucleotide detection assays of one or more varieties; a shipping component for shipping the oligonucleotide detection assays; and a billing component for billing a customer for the oligonucleotide detection assays.
  • the billing component comprises a payment receipt component for receiving payment for the oligonucleotide detection assays.
  • the computer-based customer order component comprises a client- based computer network, a physician's computer network, and insurance company computer network, a health maintenance organizations computer network, a hospital computer network, a distributor-based computer network, and/or a combination thereof.
  • the computer-based customer order component comprises a web-based user interface for ordering the oligonucleotide detection assay via single or multiple linked screens or web pages.
  • the web-based user interface provides a detection assay locator component.
  • the detection assay locator component comprises a library of detection assay data from winch an ohgonucleotide detection assay can be selected from a single type of detection assays or from a catalogue of different types of detection assays.
  • the library of detection assay data comprises single nucleotide polymorphism ("SNP") data or other data related to the SNP data.
  • the detection assay production component comprises a shop floor control system (e.g. comprising an oligonucleotide control system for synthesizing oligonucleotides, and a centralized control network for processing oligonucleotides).
  • the shop floor control system is configured to direct oligonucleotide detection assay production using a make-to-order routine, a make-to-stock routine, and/or a fulfill-from- stock routine, or other software package.
  • the shop floor control system comprises a library of detection assay data from which the plurality of detection assays of a single variety or detection assays of more than one variety can be created. It is appreciated that this library of data, the accuracy of which has been checked against a single or plurality of databases of this type of data reduces the error rates associated with detection assay production.
  • the order entry component or the billing component comprises a differential pricing component.
  • the differential pricing component is a set of routines that run on one or more processors of the system described herein.
  • the differential pricing component is capable of selectably pricing a detection assay or a single variety or a plurality of detection assays of more than one variety based upon a predetermined category of product.
  • the predetermined category of product is selected from the group consisting of an RUO product, an ASR product, and an IVD product.
  • These routines analyze the product category selection of a consumer or other purchaser to correlate the correct pricing for a detection assay with the category selected by the consumer or the end user.
  • the differential pricing component comprises a routine that associates a predetermined price of a detection assay based upon a presentation platform selection.
  • a consumer selects a 96 well plate as the detection assay presentation platform one price data set is correlated with the transaction. If the consumer selects a combination of different presentation platforms, e.g. 1536 well format, and glass slide format the routines correlate and tabulate the correct price data for the transaction.
  • presentation platforms e.g. 1536 well format, and glass slide format
  • the detection assay production component comprises a synthesis component, a cleave/deprotect component, a purification component, a dilute and fill component, and/or a quality control component.
  • the synthesis component comprises a plurality of oligonucleotide synthesizers or a single synthesizer capable of a multiplicity of syntheses. The present invention is not limited by the nature of the synthesizers.
  • Synthesizers include, but are not limited to, alone or in combination, MOSS EXPEDITE 16-channel DNA synthesizers (PE Biosystems, Foster City, CA), OligoPilot (Amersham Pharmacia,), the 3900 and 3948 48-Channel DNA synthesizers (PE Biosystems, Foster City, CA), POLYPLEX (Genemachines), 8909 EXPEDITE, Blue Hedgehog (Metabio), MerMade (BioAutomation, Piano, Texas), Polygen (Distribio, France), and PrimerStation 960 (Intelligent Bio-Instruments, Cambridge, MA).
  • the detection assay production component comprises an inventory control component.
  • the inventory control component comprises hardware, software, an optional freezer or cooler (walk in style cooler in one variant) with selectable temperature control, and robotics to place and select items of inventory in predetermined locations within the freezer, cooler or cold room.
  • the detection assay comprises an invasive cleavage assay, a TAQMAN assay, a sequencing assay, a polymerase chain reaction assay, a hybridization assay, a hybridization assay employing a probe complementary to a mutation, a microarray assay (e.g. on a solid support), a bead array assay, a primer extension assay, an enzyme mismatch cleavage assay, a branched hybridization assay, a rolling circle replication assay, a NASB A assay, a molecular beacon assay, a cycling probe assay, a ligase chain reaction assay, and a sandwich hybridization assay.
  • the detection assay is configured to detect a sequence selected comprising a polymorphism, a transgene, a splice junction, a mammalian sequence, a prokaryotic sequence, and a plant sequence. It is appreciated that one or more of these detection assays can be produced in one or more production facilities using the systems and methods of the present invention. Moreover, one ore more of these detection assays have data associated or related to each respective detection assay presented via the detection assay locator. By way of further example a particular location on the detection assay locator web page or screen can have listings for several types of detection assay for a single nucleotide polymorphism including pricing information for each respective detection assay.
  • the pricing data located thereon can be variable. For example, where there are three types of detection assay on a page, a routine automatically makes pricing for a favored or predetermined detection assay lower or competitive with one or more other types of detection assays.
  • the detection assay production component comprises an oligonucleotide detection assay design component.
  • the detection assay design component comprises a PCR primer creation component that can optionally be used alone or in combination with the detection assay design component.
  • the PCR primer creation component is configured to optimize PCR primer concentrations.
  • the detection assay design component is configured to design a single type of detection assay, a plurality of detections assays of a single variety, or a plurality of detection assays or multiple varieties for detecting the presence of one or more polymorphisms (e.g., single nucleotide polymorphisms), RNA, other sequences and/or combinations thereof.
  • the detection assay design component is configured to design a panel or array comprising a plurality of ohgonucleotide detection assays of a single variety, of multiple varieties, for a single SNP, for multiple SNPS, for a single SNP detected by multiple varieties of detection assays, and for multiple SNPs detected by multiple varieties of detection assays.
  • the detection assay production component comprises a genotyping component.
  • the genotyping component is configured to test an oligonucleotide detection assay (of a single type or multiple types) against a plurality of target sequences from different sources.
  • the present invention provides detection assay ordering systems, comprising a first processor (including one or more microprocessors) in electronic communication with: a) a computer system or single computer of a customer; b) an electronic detection assay identification catalogue going across one or more genomic landscapes; c) a second processor (including one or more microprocessors) configured to carry out detection assay design; and d) a third processor (including one or more microprocessors) configured to carry out detection assay production.
  • processors one through three can be a single processor or multiple processors located in one or more locations.
  • archival backup routines and devices provide back up for the data and routines used on one or more devices and components described herein.
  • the detection assay comprises an invasive cleavage assay or other assay described herein.
  • the first processor provides a user interface to the computer system of the customer.
  • the user interface comprises stacked databases, or linked web pages.
  • the stacked databases, screens or web pages comprise SNP data or sequence data that includes a SNP.
  • the stacked databases or web pages comprise pre-existing detection assay data.
  • the pre-existing detection assay comprises data of a detection assay that has passed through an in silico process.
  • the pre-existing detection assay data comprises data of a detection assay that has passed through a genotyping process.
  • the present invention provides systems and methods for acquiring and analyzing biological information obtained from the use of one or more types or varieties of detection assays ordered or produced using the systems and methods described herein.
  • the present invention provides systems and methods for the use of genetic information in the generation of assays for detecting the genetic identity of samples, the production of assays, the use of assays for gathering genetic information of individuals and populations, and the storage, analysis, and use of the obtained information.
  • the present invention provides a method for screening candidate oligonucleotides for use in a detection assay, comprising, providing 1) a candidate oligonucleotide, 2) five or more target nucleic acids (e.g., 6, 7, 8, . . ., 100, . .
  • each of the five or more target nucleic acids is derived from a different subject; and detection assay components that permit detection of the target nucleic acids in the presence of a functional detection oligonucleotide; treating together the five or more target nucleic acids with the candidate oligonucleotide in the presence of the detection assay components; and determining if the candidate oligonucleotide is a functional detection oligonucleotide for use with each of the five or more target nucleic acids.
  • the target nucleic acids comprise a single nucleotide polymorphism.
  • the candidate oligonucleotide comprises a hybridization probe.
  • the candidate oligonucleotide is designed to hybridize to a target sequence of at least one of the target nucleic acids.
  • the target sequence is identified by or selected by in silico analysis.
  • the detection assay components comprise detections assay components for performing an INVADER assay.
  • the method further comprises the step of preparing a kit containing the candidate oligonucleotide if the candidate oligonucleotide is determined to be a functional detection oligonucleotide.
  • the kit comprises instructions, directing a user of the kit to use the kit with samples from subjects suspected of possessing any of the target nucleic acids from which the candidate oligonucleotide was determined to be a functional detection oligonucleotide.
  • the present invention also provides a method of gathering and storing genomic data derived from a detection assay, comprising providing a detection assay configured to detect the presence or absence of a nucleic acid sequence in a sample; a first computer system comprising one or more computer processors and a computer memory; a second computer system comprising one or more computer processors and computer memory, wherein the computer memory comprises a genomic information database; and a test sample; treating the test sample with the detection assay to generate test result data; collecting the test result data with the first computer system; and transmitting the test result data from the first computer system to. the second computer system under conditions such that the test result data is added to the genomic information database of the second computer system.
  • the detection assay comprises assays including, but not limited to, hybridization assays, cleavage assays, amplification assays, sequencing assays, and ligation assays.
  • the detection comprises an INVADER assay, a TAQMAN assay, any other type of assay described herein, and or combinations thereof.
  • the nucleic acid sequence comprises a single nucleotide polymorphism or RNA.
  • the first computer system or computer including a microprocessor comprises one more detectors (e.g., fluorescent detectors, luminescent detectors, optical detectors, and radioactivity detectors).
  • the instrumentation described herein can also be sold as kit which would include the instrumentation described herein as well as a plurality of pre-ordered or ordered detection assays.
  • the test sample comprises a genomic DNA or RNA sample or a synthetic DNA or RNA sample.
  • the test sample comprises an RNA sample, and/or a PCR target/sample.
  • the test result data comprise information related to a subject from which the test sample was derived. Test result data can be presented to a user via a computer or workstation communicatively linked to any computer or display linked to any of the components described herein.
  • the first computer system (which is optionally networked) or computer is located in a different geographic location from the second computer system (which is optionally networked in a LAN, MAN, WAN, ⁇ r combination thereof) or computer.
  • the transmitting comprises sending the test result data over a communication network on which the various computers are communicatively linked, hi some preferred embodiments, the test result data comprises allele frequency information.
  • the genomic information database comprises database data comprising allele frequency information, genetic location pathway data, metabolic pathway data, and or combinations thereof.
  • the present invention further provides a method for searching nucleic acid databases comprising providing a central node comprising a processor, a plurality of sub-nodes in electronic communication with the central node, said sub-nodes comprising sequence database information, and nucleic acid sequence to be searched; providing the nucleic acid sequence to be searched to the central node; and concurrently sending the nucleic acid sequence information to be searched from the central node to the plurality of sub-nodes; and searching the sequence database information with the nucleic acid sequence to be searched to generate search results.
  • the method further comprises the step of sending the search results from the plurality of sub-nodes to the central node. In preferred embodiments, the latter steps are complete in two seconds or less.
  • two or more distinct sequence databases are stored on the plurality of sub-nodes. In some embodiments, one of the two or more distinct sequence databases is stored on two or more of the plurality of sub-nodes. In some embodiments, two or more copies of the two or more distinct sequence databases are stored on the plurality of sub-nodes. In some embodiments, each of the plurality of sub-nodes comprises a single sequence database. In some embodiments, the nucleic acid sequence to be searched comprises a single nucleotide polymorphism or RNA.
  • sequence mformation comprises one or more databases comprising GoldenPath, GenBank, dbSNP, UniGene, LocusLink, The SNP Consortium, the Japanese SNP, and HGBASE SNP, Ensemble databases.
  • the present invention also provides a system or method used in one or more components hereof for characterizing a target sequence comprising: screening the target sequence for the presence of repeat sequences and heterologous sequences to generate a masked target sequence; searching a plurality of sequence databases with the masked target sequence to generate search result data; and generating a report comprising the search result data.
  • the plurality of sequence databases comprises one or more databases including, but not limited to, polymo ⁇ hism databases, genome databases, linkage databases, and disease association databases (e.g., GoldenPath, GenBank, dbSNP, UniGene, LocusLink, and SNP Consortium databases).
  • the target sequence comprises a single nucleotide polymo ⁇ hism.
  • the report provides a reliability score, said reliability score representing a likelihood of success of detecting the target sequence performance in a detection assay.
  • the report indicates the presence or absence of the target sequence in one or more of the plurality of sequence databases.
  • the report indicates a position of the target sequence in a genome.
  • the report provides polymo ⁇ hism information related to the target sequence.
  • the present invention further provides a database (e.g. used in one or more components hereof) comprising allele frequency information, said allele frequency information generated by a method comprising: producing a detection assay for detecting a target sequence; testing five or more target sequences from different subjects with the detection assay to produce assay data; and storing the assay data in a database, wherein the assay data is correlated to at least one characteristic of the subjects.
  • the target sequence comprises a single nucleotide polymo ⁇ hism.
  • the at least one characteristic of the subjects comprises subject age, sex, race or disease state.
  • the present invention also provides a method for collecting genomic information comprising, providing: a detection assay that detects the presence of a target nucleic acid sequence in a sample, a software application on a computer system of a user, said software application configured to receive detection assay data, a database on a computer system of a service provider, a communications network, and one or more samples comprising nucleic acid; treating the one or more samples with the detection assay to generate assay data; collecting the assay data with the software application; transmitting the assay data from the computer system of the user to the computer system of the service provider using the communications network; and storing the assay data in the database.
  • the target nucleic acid sequence comprises a single nucleotide polymo ⁇ hism, wherein the detection assay detects the presence or absence of the single nucleotide polymo ⁇ hism.
  • the present invention also provides databases generated by such methods. The databases are used in one or more components hereof.
  • the present invention provides methods, systems, processes, and routines for developing and optimizing nucleic acid detection assays for use in basic research, clinical research, and for the development of clinical detection assays.
  • the present invention provides methods comprising; a) providing target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, and b) processing the target sequence information such that a primer set is generated, wherein the primer set comprises a forward and a reverse primer sequence for each of the at least Y target sequences, wherein each of the forward and reverse primer sequences comprises a nucleic acid sequence represented by 5'-N[x]-N[x-l]- ....-N[4]-N[3]-N[2]-N[l]-3', wherein N represents a nucleotide base, x is at least 6, N[l] is nucleotide A or C, andN[2]-N[l]-3' of each of the forward and reverse primers is not complementary to N[2]-N[l]
  • the present invention provides methods comprising; a) providing target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, and b) processing the target sequence information such that a primer set is generated, wherein the primer set comprises a forward and a reverse primer sequence for each of the at least Y target sequences, wherein each of the forward and reverse primer sequences comprises a nucleic acid sequence represented by 5'-N[x]-N[x-l]- ....-N[4]-N[3]-N[2]-N[l]-3', wherein N represents a nucleotide base, x is at least 6, N[l] is nucleotide G or T, and N[2]-N[l]-3' of each of the forward and reverse primers is not complementary to N[2]-N[l]
  • a method comprising; a) providing target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, and b) processing the target sequence information such that a primer set is generated, wherein the primer set comprises; i) a forward primer sequence identical to at least a portion of the 5" region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the 3' region for each of the at least Y target sequences, wherein each of the forward and reverse primer sequences comprises a nucleic acid sequence represented by 5'-N[x]-N[x-l]- ....-N[4]-N[3]-N[2]-N[l]-3', wherem N represents a nucleic acid sequence represented by 5'-N[x]-N[x
  • the present invention provides methods (including routines that provide the following functionality) comprising a) providing target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, and b) processing the target sequence information such that a primer set is generated, wherein the primer set comprises; i) a forward primer sequence identical to at least a portion of the 5' region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the 3' region for each of - the at least Y target sequences, wherein each of the forward and reverse primer sequences comprises a nucleic acid sequence represented by 5'-N[x]-N[x-l]- ....-N[4]-N[3]-N[2]-N[l]-3', wherein N represents a nucleic acid sequence
  • the present invention provides methods (and routines providing the following functionality) comprising a) providing target sequence information for at least Y target sequences, wherein each of the target sequences comprises a single nucleotide polymo ⁇ hism, b)determining where on each of the target sequences one or more assay probes would hybridize in order to detect the single nucleotide polymo ⁇ hism such that a footprint region is located on each of the target sequences, and c) processing the target sequence information such that a primer set is generated, wherein the primer set comprises; i) a forward primer sequence identical to at least a portion of the target sequence immediately 5' of the footprint region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the target sequence immediately 3' of the footprint region for each of the at least Y target sequences, wherein each of the forward and reverse primer sequences comprises a nucleic acid sequence represented by 5'-N[x]-N[x-
  • the present invention provides methods (and routines providing the following functionality) comprising a) providing target sequence information for at least Y target sequences, wherein each of the target sequences comprises a single nucleotide polymo ⁇ hism, b) determining where on each of the target sequences one or more assay probes would hybridize in order to detect the single nucleotide polymo ⁇ hism such that a footprint region is located on each of the target sequences, and c) processing the target sequence information such that a primer set is generated, wherein the primer set comprises; i) a forward primer sequence identical to at least a portion of the target sequence immediately 5 ' of the footprint region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the target sequence immediately 3 ' of the footprint region for each of the at least Y target sequences, wherein each of the forward and reverse primer sequences comprises a nucleic acid sequence represented by 5'-N[x]-N
  • the primer set is configured for performing a multiplex PCR reaction that amplifies at least Y amplicons, wherein each of the amplicons is defined by the position of the forward and reverse primers.
  • the primer set is generated as digital or printed sequence information.
  • the primer set is generated as physical primer oligonucleotides. Using the methods, routines and components herein is it possible to generate 100-plex and greater PCR primer reactions.
  • N[3]-N[2]-N[l]-3' of each of the forward and reverse primers is not complementary to N[3]-N[2]-N[l]-3' of any of the forward and reverse primers in the primer set.
  • the processing comprises initially selecting N[l] for each of the forward primers as the most 3' A or C in the 5' region. In certain embodiments, the processing comprises initially selecting N[l] for each of the forward primers as the most 3' G or T in the 5' region. In some embodiments, the processing comprises initially selecting N[l] for each of the forward primers as the most 3' A or C in the 5' region, and wherein the processing further comprises changing the N[l] to the next most 3' A or C in the 5' region for the forward primer sequences that fail the requirement that each of the forward primer's N[2]-N[l]-3' is not complementary to N[2]-N[l]-3' of any of the forward and reverse primers in the primer set. In other embodiments, the processing (preferably electronic) comprises initially selecting
  • the processing comprises initially selecting N[l] for each of the reverse primers as the most 3' G or T in the complement of the 3' region. In further embodiments, the processing comprises initially selecting N[l] for each of the reverse primers as the most 3' A or C in the 3' region, and wherein the processing further comprises changing the N[l] to the next most 3' A or C in the 3' region for the reverse primer sequences that fail the requirement that each of the reverse primer's N[2]-N[l]-3' is not complementary to N[2]-N[l]-3' of any of the forward and reverse primers in the primer set.
  • the footprint region comprises a single nucleotide polymo ⁇ hism.
  • the footprint comprises a mutation.
  • the footprint region for each of the target sequences comprises a portion of the target sequence that hybridizes to one or more assay probes configured to detect the single nucleotide polymo ⁇ hism.
  • the footprint is this region where the probes hybridize.
  • the footprint further includes additional nucleotides on either end.
  • the processing further comprises selecting N[5]-N[4]-N[3]-N[2]-N[l]-3',for each of the forward and reverse primers such that less than 80 percent homology with a assay component sequence is present.
  • the assay component is a FRET probe sequence.
  • the target sequence is about 300-500 base pairs in length, or about 200-600 base pair in length.
  • Y is an integer between 2 and 500, or between 2-10,000.
  • the processing comprises selecting x for each of the forward and reverse primers such that each of the forward and reverse primers has a melting temperature with respect to the target sequence of approximately 50 degrees Celsius (e.g. 50 degrees, Celsius, or at least 50 degrees Celsius, and no more than 55 degrees Celsius).
  • the melting temperature of a primer is at least 50 degrees Celsius, but at least 10 degrees different than a selected detection assay's optimal reaction temperature.
  • the forward and reverse primer pair optimized concentrations are determined for the primer set.
  • the processing is automated. In further embodiments, the processing is automated with a processor.
  • the present invention provides a kit comprising the primer set generated by the methods of the present invention, and at least one other component (e.g. cleavage agent, polymerase, INVADER oligonucleotide, or other detection assay or detection assay component in another variant of the invention).
  • the present invention provides compositions comprising the primers and primer sets generated by the methods of the present invention.
  • the present invention provides methods (and routines utilizing methodology) comprising; a) providing; i) a user interface configured to receive sequence data, ii) a computer system having stored therein a multiplex PCR primer software application, and b) transmitting the sequence data from the user interface to the computer system, wherein the sequence data comprises target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, and c) processing the target sequence information with the multiplex PCR primer pair software application to generate a primer set, wherein the primer set comprises; i) a forward primer sequence identical to at least a portion of the target sequence immediately 5' of the footprint region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the target sequence immediately 3' of the footprint region for each of
  • the present invention provides methods (and routines used in the methodology) comprising; a) providing; i) a user interface configured to receive sequence data, ii) a computer system having stored therein a multiplex PCR primer software application, and b) transmitting the sequence data from the user interface to the computer system, wherein the sequence data comprises target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, and c) processing the target sequence information with the multiplex PCR primer pair software application to generate a primer set, wherein the primer set comprises; i) a forward primer sequence identical to at least a portion of the target sequence immediately 5' of the footprint region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the target sequence immediately 3' of the footprint region for each
  • the present invention provides systems comprising; a) a computer system (and routines used in the methodology) configured to receive data from a user interface, wherein the user interface is configured to receive sequence data, wherein the sequence data comprises target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, b) a multiplex PCR primer pair software application operably linked to the user interface, wherein the multiplex PCR primer software application is configured to process the target sequence information to generate a primer set, wherein the primer set comprises; i) a forward primer sequence identical to at least a portion of the target sequence immediately 5' of the footprint region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the target sequence immediately 3' of the footprint region for each of the at least Y target sequences
  • the present invention provides systems comprising; a) a computer system or computer configured to receive data from a user interface, wherein the user interface is configured to receive sequence data, wherein the sequence data comprises target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, b) a multiplex PCR primer pair software application operably linked to the user interface, wherein the multiplex PCR primer software application is configured to process the target sequence information to generate a primer set, wherein the primer set comprises; i) a forward primer sequence identical to at least a portion of the target sequence immediately 5' of the footprint region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the target sequence immediately 3' of the footprint region for each of the at least Y target sequences, wherein each of the forward primer sequence identical to
  • the present invention relates to novel methods of producing oligonucleotides.
  • the present invention provides an efficient, safe, and automated process for the production of large quantities of oligonucleotides.
  • the present invention provides high-throughput oligonucleotide production systems comprising: an oligonucleotide synthesizer component, wherein the oligonucleotide synthesizer component comprises at least 100 oligonucleotide synthesizers.
  • the system further comprises at least one oligonucleotide processing component.
  • the system further comprises a centralized control network operably linked to the oligonucleotide synthesizer component.
  • the present invention provides methods for the high throughput production of oligonucleotides comprising; a) providing an oligonucleotide synthesizer component; and b) generating a high through-put quantity of oligonucleotides with the oligonucleotide synthesizer component, wherein the high through-put quantity comprises at least 1 per hour (e.g. at least 1, 10, 100, 1000, etc, per hour).
  • the present invention provides methods for the production of an oligonucleotide comprising: a) providing; i) a first computer memory device comprising oligonucleotide specification information, and ii) an oligonucleotide synthesizer component, wherein the oligonucleotide synthesizer component comprises a) at least 100 oligonucleotide synthesizers (in another variant the number of synthesizers can be in the range of about 20 to about 1000 synthesizers depending on the number of syntheses each synthesizer is capable of executing), and b) a second computer memory device; and b) conveying the oligonucleotide specification information from the first computer memory device to the second computer memory device under conditions such that the oligonucleotide synthesizer component generates at least one oligonucleotide (e.g. at least 1, 10, 100, 1000, etc).
  • high throughput synthes e.g. at least 1, 10,
  • the present invention provides oligonucleotide production systems comprising: a) an oligonucleotide production component configured for divergent production of a set of oligonucleotides, wherein the set of oligonucleotides comprises first and second corresponding oligonucleotides, and wherein the oligonucleotide production component comprises first and second oligonucleotide manufacturing components; and b) a centralized control network operably linked to the oligonucleotide production component, wherein the centralized control network is configured for controlling the divergent production of the set of oligonucleotides.
  • the present invention provides methods for the divergent production of oligonucleotides comprising; a) providing an oligonucleotide production component comprising an oligonucleotide synthesizer component and at least one oligonucleotide processing component; and b) employing the oligonucleotide production component for divergent production of a set of oligonucleotides, wherein the set of oligonucleotides comprises first and second corresponding oligonucleotides.
  • the present invention provides high-throughput oligonucleotide purification systems comprising a plurality of HPLC devices operably connected to a single sample injector.
  • the system further comprises a centralized control network.
  • the present invention provides methods for the high- throughput purification of oligonucleotides comprising: a) providing; i) an oligonucleotide purification component comprising a plurality of HPLC devices operably connected to a single sample injector, and ii) an oligonucleotide sample comprising full-length oligonucleotides and truncated oligonucleotides; and b) processing the sample with the oligonucleotide purification component under conditions such that at least a portion of the truncated oligonucleotides are removed from the oligonucleotide sample.
  • the present invention provides high-throughput oligonucleotide production systems comprising; a) an oligonucleotide production component comprising first and second oligonucleotide manufacturing components; and b) a sample rack configured for use in the first and second oligonucleotide manufacturing components without modification.
  • the system further comprises a central reagent supply network.
  • the present invention provides methods for high-throughput processing of oligonucleotide samples, comprising: a) providing; i) an oligonucleotide production component comprising first and second manufacturing components, and ii) a sample rack integrated with the first manufacturing component, wherein the sample rack is configured for use in the first and second oligonucleotide manufacturing components without modification, and wherein the sample rack comprises a plurality of oligonucleotide samples; and b) processing at least a portion of the plurality of oligonucleotide samples with the first manufacturing component, c) transferring the sample rack from the first manufacturing component to the second manufacturing component; and d) processing at least a portion of the oligonucleotide samples with the second manufacturing component.
  • the present invention provides high-throughput oligonucleotide dry-down systems comprising a centrifugal evaporator configured for processing at least 1 aqueous oligonucleotide sample in one hour or less.
  • the system is configured for processing at least 5 oligonucleotide samples per hour (e.g. 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50).
  • the present invention provides high-throughput oligonucleotide dry down systems comprising a centrifugal evaporator configured for processing a plurality of oligonucleotide samples in one hour or less, wherein the plurality of oligonucleotide samples comprises at least 1 liter of water (e.g. 1, 5, 10, 15, 35 or 50 liters of water).
  • a centrifugal evaporator configured for processing a plurality of oligonucleotide samples in one hour or less, wherein the plurality of oligonucleotide samples comprises at least 1 liter of water (e.g. 1, 5, 10, 15, 35 or 50 liters of water).
  • the present invention provides methods for the high-throughput dry-down of oligonucleotides comprising: a) providing; i) an oligonucleotide dry-down component comprising a centrifugal evaporator, and ii) a plurality of oligonucleotide samples comprising at least 10 aqueous oligonucleotide samples; and b) processing the plurality of oligonucleotide samples with the oligonucleotide dry-down component, wherem the processing renders each of the aqueous oligonucleotide samples substantially water-free in one hour or less.
  • the present invention provides methods for the high-throughput dry-down of oligonucleotides comprising: a) providing; i) an oligonucleotide dry-down component comprising a centrifugal evaporator, and ii) a plurality of aqueous oligonucleotide samples, wherein the plurality of oligonucleotide samples comprises at least one liter of water, and b) processing the plurality of oligonucleotide samples with the oligonucleotide dry-down component, wherein the processing renders the plurality of aqueous oligonucleotide samples substantially water-free in one hour or less.
  • the present invention provides high-throughput oligonucleotide de-salting systems comprising an oligonucleotide de-salting component configured for processing at least 150 oligonucleotide samples per half hour.
  • the oligonucleotide de-salting component comprises a robotic oligonucleotide sample handling device, and a sample rack.
  • the present invention provides methods for the high-throughput de-salting of oligonucleotides comprising: a) providing; i) an oligonucleotide de-salting component comprising a robotic oligonucleotide sample handling device, and ii) a plurality of oligonucleotide samples comprising at least 150 oligonucleotide samples; andb) processing the plurality of oligonucleotide samples with the oligonucleotide de-salting component, wherein the processing renders each of the oligonucleotide samples substantially salt-free in a half-hour or less.
  • the present invention provides high-throughput oligonucleotide dilute and fill systems comprising an oligonucleotide dilute and fill component, wherein the ohgonucleotide dilute and fill component comprises an automated liquid processing device operably linked to a spectrophotometer.
  • the present invention provides methods method for the high- throughput dilute and fill of oligonucleotide samples comprising: a) providing; i) an oligonucleotide dilute and fill component comprising an automated liquid processing device operably linked to a spectrophotometer, and ii) a plurality of oligonucleotide samples; and b) processing the plurality of oligonucleotide samples with the oligonucleotide dilute and fill component, wherein the processing normalizes each of the oligonucleotide samples.
  • concentration is an important aspect of the invention with respect to the production of detection assays.
  • oligonucleotide production samples have their concentrations normalized. This normalization can be accomplished via the utilization of known extinction coefficient methods and knowledge of the sequence from production information.
  • the present invention also provides a nucleic acid synthesis reagent delivery system comprising: one or more reagent containers containing nucleic acid synthesis reagent; a branched delivery component attached to said one or more reagent containers such that the nucleic acid synthesis reagent can pass from said reagent containers to said branched delivery component, wherein the branched delivery component comprises a plurality of branches; and a plurality of delivery lines, the plurality of delivery lines attached on one end to a branch of the branched delivery component and attached on a second end to a nucleic acid synthesizer.
  • the present invention is not limited by the number branches or delivery lines.
  • the plurality of branches comprises ten or more branches.
  • the plurality of delivery lines comprises ten or more delivery lines.
  • the branched delivery component comprises a sight glass.
  • the sight glass comprises a purge valve.
  • the one or more of the plurality of delivery lines comprises a shut-off valve.
  • the present invention further provides a waste disposal system comprising: a waste tank comprising a waste input channel configured to receive liquid waste product and a waste output channel configured to remove liquid waste when the waste tank is purged; and a pressurized gas line attached to the waste tank, the pressurized gas line configured to deliver gas into the waste tank when the waste tank is to be purged, wherein the gas line is configured to deliver a gas that allows purging of the waste tank.
  • the pressurized gas line is attached to an argon gas source.
  • the gas is delivered at a low pressure (e.g., 3-10 pounds per square inch).
  • the waste input channel is attached to a waste line, wherein the waste line is attached to a plurality of nucleic acid synthesizers (e.g., 20 or more nucleic acid synthesizers).
  • the waste tank comprises a sight glass.
  • the system further comprises an automated purge component, said automated purge component capable of detecting waste levels in the waste tank and purging the waste tank when the waste levels are at or above a threshold level (e.g., a pre- selected threshold level) .
  • the present invention also provides a method for purifying nucleic acids comprising providing: an nucleic acid purification column, a buffer, and a nucleic acid mixture; contacting the nucleic acid mixture with the nucleic acid purification column; and adding the buffer to the nucleic acid purification column, wherein a nucleic acid molecule having between 23-39 nucleotides is eluted from the nucleic acid purification column in less than forty minutes, and in one variant of the invention can be accomplished in less than about 25 minutes.
  • the nucleic acid purification column is contained in an HPLC apparatus.
  • the present invention further provides a method for deprotecting nucleic acid molecules comprising providing: a multiwell plate configured to hold a plurality of protected nucleic acid molecules and a plurality of different protected nucleic acid molecules; placing the nucleic acid molecules into the multiwell plates; and treating the plate under conditions that resulted in the deprotection of the nucleic acid molecules.
  • the multiwell plate comprises a 96-well plate.
  • the present invention relates to nucleic acid synthesizers and methods of using and modifying nucleic acid synthesizers.
  • the present invention provides highly efficient, reliable, and safe synthesizers that find use, for example, in high throughput and automated nucleic acid synthesis, as well as methods of modifying pre-existing synthesizers to improve efficiency, reliability, and safety.
  • the present invention also relates to synthesizer arrays for efficient, safe, and automated processes for the production of large quantities of oligonucleotides.
  • the present invention provides systems comprising a synthesis and purge component, the synthesis and purge component comprising a cartridge and a drain plate, wherein the cartridge is configured to hold one or more nucleic acid synthesis columns and wherein the cartridge is separated from the drain plate by a drain plate gasket.
  • the cartridge is configured to hold a plurality of nucleic acid synthesis columns.
  • the cartridge is configured to hold 12 or more nucleic acid synthesis columns.
  • the cartridge is configured to hold 48 or more nucleic acid synthesis columns.
  • the cartridge is configured to hold exactly 48 nucleic acid synthesis columns.
  • the assembly comprising the cartridge, the drain plate and the drain plate gasket is configured to provide a substantially airtight seal between the assembly and the outside of each nucleic acid synthesis column.
  • the airtight seal between the assembly and each column is provided by an O-ring.
  • each O-ring is positioned between the cartridge and the exterior surface of a column.
  • any material that provides a compressible interface can be used in the invention.
  • the drain plate gasket provides a substantially airtight seal between the cartridge and the drain plate. In other embodiments, the drain plate gasket provides an airtight seal between the cartridge and the drain plate. In some embodiments, the drain plate gasket comprises one or more alignment markers configured to allow aligned attachment of said cartridge to said drain plate. In additional embodiments, the drain plate gasket comprises one or more alignment markers configured to allow aligned attachment of the drain plate gasket to the cartridge. In other embodiments, the drain plate gasket comprises one or more alignment markers configured to allow aligned attachment of the gasket to the drain plate. In certain embodiments, the drain plate gasket comprises at least one drain cut-out. In other embodiments, the drain plate gasket comprises at least four drain cut-outs.
  • the drain plate gasket comprises one drain cut out for every synthesis column in the cartridge.
  • the cut outs in the drain plate gasket for each synthesis column are configured to provide an airtight seal between the outside of each nucleic acid synthesis column and the assembly comprising the cartridge, the drain plate, and the drain plate gasket.
  • the present invention provides systems comprising a synthesis and purge component, the synthesis and purge component comprising a cartridge and a drain plate, wherein the cartridge is configured to hold one or more nucleic acid synthesis columns and wherein the cartridge is separated from the drain plate by a drain plate gasket.
  • the drain plate comprises at least one drain (e.g. 1, 2, 3, 4, 5, 10,... 20, ).
  • the system further comprises a waste tube, the waste tube comprising input and output ends, wherein the input end is configured to receive waste materials from the drain.
  • the waste tube comprises an inner diameter of at least 0.187 inches (preferably at least 0.25 inches).
  • the waste tube and the drain are configured such that, when the drain is contacted with the waste tube for waste removal, the waste tube encloses at least a portion of the drain (See, e.g., Figure 40).
  • the drain forms a sealed contact point with an interior portion of the waste tube when the drain is enclosed in the waste tube.
  • the drain further comprises a drain sealing ring.
  • the system further comprises a waste valve wherein the waste valve is configured to receive waste from the output end of the waste tube.
  • the waste valve comprises an interior diameter of at least 0.187 inches (preferably at least 0.25 inches).
  • the waste valve provides a straight-through path for the waste (e.g. as opposed to an angled path). Straight-through paths can be accomplished, for example, by the use of a gate or ball valve.
  • the system further comprises a plurality of dispense lines, the dispense line configured for delivering at least one reagent to a synthesis column in the cartridge.
  • the dispense lines comprise an interior diameter of at least 0.25 mm.
  • the system further comprises an alignment detector.
  • the alignment detector is configured to detect the alignment of a waste tube and a drain. In other embodiments, the alignment detector is configured to detect the alignment of a dispense line and a receiving hole of the cartridge. In some embodiments, the alignment detector is configured to detect a tilt alignment of the synthesis and purge component.
  • the system of the present invention further comprises a motor attached to the synthesis and purge component and configured to rotate the synthesis and purge component.
  • the motor is attached to the synthesis and purge component by a motor connector.
  • the system further comprises a bottom chamber seal positioned between the motor connector and the synthesis and purge component.
  • the system of the present invention comprises two drain. In preferred embodiments, the two drain are located on opposite sides of the drain plate.
  • the synthesis and purge component is contained in a chamber.
  • a chamber bowl and a top cover combine to form a chamber (e.g.
  • the chamber comprises a bottom surface (e.g. bottom of a chamber bowl, see, e.g. Figure 41) comprising the top portion of two waste tubes (which may, for example, extend downward from bottom of the chamber).
  • the waste tubes are positioned symmetrically on the bottom surface of the chamber (see, e.g., Figure 41).
  • the systems of the present invention further comprise a chamber drain having open and closed positions, the chamber drain configured to allow gas emissions (or liquid waste) to pass out of the chamber when in the open position.
  • the systems of the present invention further comprise a reagent dispensing station, wherein the reagent dispensing station is configured to house one or more reagent reservoirs, such that reagents in reagent reservoirs can be delivered to the cartridge.
  • the reagent dispensing station comprises one or more ventilation tubes
  • the reagent dispensing station provides an enclosure.
  • the enclosure comprises a viewing window to allow visual inspection of the reagent reservoirs without opening the enclosure.
  • one reagent dispensing station is configured to serve multiple synthesizers.
  • the systems of the present invention are capable of maintaining a gas pressure in the chamber sufficient to purge synthesis columns prior to addition of reagents to the synthesis columns.
  • the nucleic acid synthesis systems of the present invention comprise a cartridge in a chamber, the cartridge comprising a plurality of synthesis columns, wherein the synthesis columns contain packing material that provides a resistance against pressurized gas contained in the chamber, the resistance being sufficient to maintain a pressure in the chamber that is capable of purging synthesis columns prior to addition of reagents to the synthesis columns.
  • one or more of the plurality of synthesis columns does not undergo a synthesis reaction.
  • the packing material comprises a frit.
  • the frit is a bottom frit.
  • the frit is a top frit.
  • the packing material comprises a top frit, solid support, and a bottom frit.
  • the solid support is polystyrene.
  • the packing material comprises a synthesis matrix.
  • the present invention provides nucleic acid synthesis systems comprising a synthesis and purge component in a pressurized chamber, the synthesis and purge component comprising a plurality of synthesis columns, wherein the synthesis columns contain packing material sufficient to maintain pressure in the chamber during a purging operation to purge liquid reagent from the plurality of synthesis columns when at least one of the plurality of synthesis columns does not contain liquid reagent.
  • more than one of the plurality of synthesis columns e.g. 2, 3, 5, 10) do not contain liquid reagent (and the remaining synthesis columns do contain liquid reagent).
  • the present invention provides nucleic acid synthesis systems comprising: a) a synthesis and purge component, the synthesis and purge component comprising a cartridge and a drain plate separated by a drain plate gasket, wherein the cartridge is configured to hold twelve or more nucleic acid synthesis columns; b) a drain positioned in the drain plate; c) a chamber comprising an inner surface, the chamber housing the synthesis and purge component and the drain; d) a waste tube, the waste tube comprising input and output ends, wherein the input end is configured to receive waste materials from the drain, wherein the waste tube comprises an inner diameter of at least 0.187 inches; e) a waste valve configured to receive waste from the output end of the waste tube, wherein the waste valve comprises in interior diameter of at least 0.187 inches; f) a reagent dispensing station, wherein the reagent dispensing station is configured to house one or more reagent reservoirs; g) a plurality of dispense lines, the dispense lines configured
  • the system is capable of maintaining gas pressure in the chamber at a sufficient level to purge the synthesis columns prior to addition of reagents to the synthesis columns.
  • the synthesizer further comprises providing energy, such as heat, to the synthesis columns. Heating of the synthesis column finds use, for example, in decreasing the coupling time during a nucleic acid synthesis. It can also broaden the range of the chemical protocols that can be used in high throughput synthesis, e.g. by improving the efficiency of less efficient chemistries, such as the phosphate triester method of oligonucleotide synthesis.
  • the synthesizer further comprises a mixing component, such as an agitator, configured to agitate the synthesis columns (e.g., to mix reaction components, and to facilitate mass exchange between the reaction medium and the solid support).
  • the present invention provides methods for synthesizing nucleic acids comprising: a) providing: i) a nucleic acid synthesizer comprising a synthesis and purge component, the synthesis and purge component comprising a cartridge and a drain plate, wherein the cartridge holds a plurality of nucleic acid synthesis columns and wherein the cartridge is separated by a drain plate gasket from the drain plate, and ii) nucleic acid synthesis reagents; and b) introducing a portion of the nucleic acid synthesis reagents into at least one of the nucleic acid synthesis columns to provide a first synthesis reaction; c) purging the nucleic acid synthesis columns by creating a pressure differential across the nucleic acid synthesis columns; and d) introducing a second portion of the nucleic acid synthesis reagents into at least one of the nucleic acid synthesis columns to provide a second synthesis reaction.
  • the drain plate gasket provides a substantially airtight seal between the cartridge and the drain plate. In other embodiments, the drain plate gasket provides an airtight seal between the cartridge and the drain plate.
  • the present invention further provides a cartridge for use in an open nucleic acid synthesis system, said cartridge comprising a plurality of receiving holes configured to hold nucleic acid synthesis columns, wherein the cartridge is further configured to receive one or more O-rings, wherein the presence of the one or more O-rings provides a seal between the nucleic acid synthesis columns and the plurality of receiving holes (i.e., the O-ring contacts an interior wall of the receiving hole and an exterior wall of the synthesis column to form a seal).
  • the cartridge is provided as part of a nucleic acid synthesis system.
  • the present invention is not limited by the nature of the O-ring.
  • the cartridge is associated with a gasket, wherein the gasket provides the O-rings (e.g., through one or more holes in the gaskets, such that when the gasket is associated with the cartridge [e.g., affixed to an outer surface of the cartridge] a seal is formed between the a receiving hole of the cartridge and a synthesis column within the receiving hole [see e.g., Figure 46C]).
  • the O-ring is provided in a groove within the receiving hole.
  • the groove is located at the top surface of the receiving hole.
  • the plurality of receiving holes comprise an upper portion and a lower portion, wherein the lower portion comprises a first diameter and the upper portion comprises a second diameter that is larger than the first diameter (see e.g., Figure 46A).
  • the groove is located within an interior portion of the receiving hole.
  • the plurality of receiving holes comprise an upper portion with a first diameter, a middle portion with a second diameter, and a lower portion with a third diameter, wherein the second diameter is larger than the first diameter and larger than the third diameter (the first and third diameters may be the same as each other or different).
  • the O-ring When an O-ring is placed in the groove, the O-ring contains an internal diameter less than the first diameter and less than the third diameter, such that it can contact a synthesis column placed within the receiving hole (see e.g., Figure 46B).
  • the cartridge comprises a rotary cartridge.
  • O-rings are provided in the cartridge.
  • the O-ring is configured to form a substantially airtight or pressure-tight seal between the receiving hole and the nucleic acid synthesis column, when said nucleic acid synthesis column is present.
  • the present invention further provides a nucleic acid synthesis system comprising a synthesis and purge component in a pressurizable chamber, said synthesis and purge component comprising a cartridge, wherein the cartridge in configured to hold a plurality of nucleic acid synthesis columns, and wherem said cartridge is further configured to provides seals between said cartridge and each of said plurality of nucleic acid synthesis columns so as to maintain pressure in said chamber during a purging operation to purge liquid reagent from said plurality of synthesis columns.
  • each of the seals between the cartridge and the plurality of nucleic acid synthesis columns is provided by an O-ring.
  • the present invention provides a nucleic acid synthesizer comprising a plurality of synthesis columns and an energy input component that imparts energy to said plurahty of synthesis columns to increase nucleic acid synthesis reaction rate in said plurality of synthesis columns.
  • said energy input component comprises a heating component.
  • said heating component provides substantially uniform heat.
  • said energy input component provides heated reagent solutions to said plurality of synthesis columns.
  • said energy input component comprises a heating coil.
  • said energy input component comprises a heat blanket.
  • said heating component comprises a resistance heater, a Peltier device, a magnetic induction device or a microwave device.
  • said energy input component comprises a heated room. In further embodiments, said energy input component provides energy in the electromagnetic spectrum. In yet other embodiments, said energy input component comprises an oscillating member. In some embodiments, said energy input component provides a periodic energy input, and in other embodiments, said energy input component provides a constant energy input.
  • said energy input heats said plurality of synthesis columns in the range of about 20 to about 60 degrees Celsius.
  • the present invention provides a nucleic acid synthesizer comprising a fail-safe reagent delivery component configured to deliver one or more reagent solutions to said plurality of synthesis columns.
  • the fail-safe reagent delivery component comprises a plurality of reagent tanks.
  • said plurality of reagent tanks comprise one or more tanks selected from the group consisting of acetonitrile tanks, phosphoramidite tanks, argon gas tanks, oxidizer tanks, tetrazole tanks, and capping solution tanks.
  • said reagent tanks comprise a plurality of large volume containers, each said large volume container comprising at least one of said reagent solutions.
  • the present invention provides high-throughput oligonucleotide production systems comprising: an oligonucleotide synthesizer anay, wherein the oligonucleotide synthesizer array comprises at least 5 oligonucleotide synthesizers.
  • the oligonucleotide synthesizer anay comprises at least 10 or at least 100 oligonucleotide synthesizers.
  • the system further comprises a centralized control network operably linked to the oligonucleotide synthesizer component.
  • the present invention provides methods for the high throughput production of oligonucleotides comprising; a) providing an oligonucleotide synthesizer array; and b) generating a high through-put quantity of oligonucleotides with the oligonucleotide synthesizer array, wherein the high through-put quantity comprises at least 1 per hour (e.g. at least 1 , 10, 100, 1000, etc, per hour).
  • the present invention provides a production facility comprising an anay of synthesizers.
  • the production facility of the present invention comprises a fail-safe reagent delivery system.
  • the production facility of the present invention comprises a centralized waste collection system.
  • the production facility of the present invention comprises a centralized control system.
  • the production facility of the present invention comprises a fail-safe reagent delivery system, a centralized waste collection system and a centralized control system.
  • the present invention provides an automated production process.
  • the automated production process includes an oligonucleotide synthesizer component and an oligonucleotide-processing component.
  • the present invention also provides integrated systems that link nucleic acid synthesizers to other nucleic acid production components.
  • the present invention provides a system comprising a nucleic acid synthesizer and a cleavage and deprotect component.
  • the synthesizer is configured for parallel synthesis of nucleic acid molecules in three or more synthesis columns.
  • the system further comprises sample tracking software configured to associate sample identification tags (e.g., electronic identification numbers, barcodes) with samples that are processed by the nucleic acid synthesizer and the cleavage and deprotect component.
  • sample tracking software is further configured to receive synthesis request information from a user, prior to sample processing by the nucleic acid synthesizer.
  • the system further comprises a robotic component configured to transfer columns from the nucleic acid synthesizer to the cleavage and deprotect component.
  • the robotic component is further configured to transfer the columns from the cleavage and deprotect component to a purification component and/or to additional production components described herein.
  • the present invention also provides control systems for operating one or more components of the systems of the present invention.
  • the present invention provides a system comprising a processor, wherein the processor is configured to operate a nucleic acid synthesizer for parallel synthesis of three or more nucleic acid molecules.
  • the present invention further provides a system comprising a processor, wherein said processor is configured to operate a nucleic synthesizer and a cleavage and deprotect component.
  • the system further comprises a computer memory, wherein the computer memory comprises nucleic acid sample order information (e.g., information obtained from a user specifying the identity of a polymer to be synthesized and/or specifying one or more characteristics of the polymer such as sequence information).
  • the computer memory further comprises allele frequency information and/or disease association information.
  • the present invention provides oligonucleotide synthesizers comprising a reaction chamber and a lid, wherein in an open position, the lid provides a substantially enclosed ventilated workspace.
  • the present invention provides methods of protecting an operator of an oligonucleotide synthesizer comprising channeling ambient air away from an operator toward an interior space of the synthesizer (e.g. down through the top surface, or up through the top cover).
  • the present invention provides apparatuses comprising, in combination, an oligonucleotide synthesizer and a venting hood.
  • the apparatuses are for production of oligonucleotides, wherein the apparatus comprises a venting component configured to draw air away from a reaction chamber of the apparatus.
  • the present invention provides systems comprises a plurality of oligonucleotide apparatuses (e.g. e.g. at least 100 synthesizers).
  • the present invention provides a polymer synthesizer comprising a ventilated workspace.
  • the polymer synthesizer is a nucleic acid synthesizer.
  • the synthesizer comprises a top enclosure, wherein the top enclosure comprises a top plate with a ventilation opening, wherein the top enclosure is configured for attachment to a top cover of a synthesizer to form a primarily enclosed space over the top cover.
  • the synthesizer comprises a base, wherein the base comprises a primarily enclosed space and a ventilation opening.
  • the top plate is configured for attachment to a ventilation tube such that air in the primarily enclosed space may be drawn through the ventilation opening into the ventilation tube.
  • the top plate further comprises an outer window, and wherein the ventilation opening is formed in the outer window.
  • the top enclosure further comprises at least four sides (e.g. 4 sides, 5 sides, etc.).
  • the top cover further comprises a ventilation slot.
  • the present invention provides polymer synthesizer (e.g. nucleic acid synthesizer) comprising; a) a top cover with a ventilation slot, and b) a top enclosure, wherein the top enclosure comprises a top plate with a ventilation opening, and wherein the top enclosure is attached to the top cover to form a primarily enclosed space above the top cover.
  • polymer synthesizer e.g. nucleic acid synthesizer
  • the present invention provides a lid enclosure comprising; a) a top cover with a ventilation slot, and b) a top enclosure, wherein the top enclosure comprises a top plate with a ventilation opening, and wherein the top enclosure is attached to the top cover to form a primarily enclosed space over the top cover.
  • the top plate is configured for attachment to a ventilation tube.
  • the top plate is configured for attachment to a ventilation tube such that air in the primarily enclosed space may be drawn through the ventilation opening into the ventilation tube.
  • the top cover is configured to attach to a top surface of a nucleic acid synthesizer with a chamber bowl.
  • the ventilation slot is configured such that air in the chamber bowl may drawn in through the ventilation slot and into the primarily enclosed space.
  • the top plate further comprises an outer window, and wherein the ventilation opening is formed in the outer window.
  • the top enclosure further comprises at least four sides.
  • the present invention provides a polymer synthesizer (e.g., nucleic acid synthesizer) comprising; a) a top surface of a nucleic acid synthesizer, b) a lid enclosure comprising; i) a top plate with a ventilation opening, and ii) a top cover with a ventilation slot; and wherein the lid enclosure is attached to the top surface.
  • the lid enclosure is attached to the top surface by at least one hinge such that the lid enclosure may be raised and lowered.
  • the present invention provides systems comprises a plurality of the polymer synthesizers (e.g., at least 100 synthesizers).
  • the present invention provides side panels configured to extend between at least one side of a top cover (or lid enclosure) and a top surface of a nucleic acid synthesizer such that a barrier to air is created on at least one side of the synthesizer when the top cover is extended upward from the top surface.
  • the present invention provides a panel (e.g. front panel or side panel) configured to extend at least part way between at least one side of a top cover (or lid enclosure) and a top surface of a nucleic acid synthesizer such that at least a partial barrier to air is created on at least one side of the synthesizer when the top cover is extended upward such that it is not in contact with the top surface.
  • the present invention provides polymer synthesizers (e.g. nucleic acid synthesizers) summary comprising; a) a top surface of a nucleic acid synthesizer, b) a lid enclosure comprising; i) a top plate with a ventilation opening, ii) a top cover with a ventilation slot; and iii) at least one top enclosure side; and c) a panel; wherein the lid enclosure is attached to the top surface by at least one hinge such that the lid enclosure may be raised and lowered, and wherein the panel is configured to extend (at least part way) between the at least one top enclosure side and the top surface such that at least a partial barrier to air is created when the lid enclosure is extended upward from the top surface.
  • the present invention provides systems comprising a plurality of the polymer synthesizers (e.g., at least 100 synthesizers).
  • the present invention provides systems comprising; a) a ventilation tube, and b) a lid enclosure comprising; a) a top cover with a ventilation slot, and b) a top enclosure comprising a top plate with a ventilation opening, wherein the top enclosure is attached to the top cover to form a primarily enclosed space over the top cover.
  • the systems further comprise a vacuum source (e.g. centralized vacuum system).
  • the top plate is configured for attachment to the ventilation tube.
  • the ventilation tube is configured for attachment to the vacuum source.
  • the system further comprises a synthesis and purge component, the synthesis and purge component comprising a cartridge and a drain plate separated by a drain plate gasket, wherein the cartridge is configured to hold a plurality of nucleic acid synthesis columns.
  • the systems further comprise a plurality of dispense lines, wherein the plurality of dispense lines are located in the primarily enclosed space.
  • the systems further comprise at least one side panel, wherein the at least one side panel is configured to extend between at least one side of the lid enclosure and a top surface of a nucleic acid synthesizer (e.g., such that a barrier to air is created on at least one side of the synthesizer when the top cover is extended upward from the top surface).
  • a nucleic acid synthesizer e.g., such that a barrier to air is created on at least one side of the synthesizer when the top cover is extended upward from the top surface.
  • the present invention provides systems comprising; a) a nucleic acid synthesizer comprising; i) a top surface, and ii) a top cover comprising a ventilation slot, wherein the top cover is attached to the top surface by at least one hinge such that the top surface may be raised and lowered; and b) a panel configured to extend at least part way between at least one side of the top cover and the top surface such that at least a partial barrier to air is created on at least one side of the nucleic acid synthesizer when the top cover is extended upward.
  • the panel is configured to fully extend between the at least one side of the top cover and the top surface such that a complete barrier to air is created on at least one side of the nucleic acid synthesizer when the top cover is extended upward.
  • the panel comprises a side panel or a front panel.
  • the system further comprises a top enclosure, wherein the top enclosure comprises a top plate with a ventilation opening, and wherein the top enclosure is attached to the top cover to form a primarily enclosed space over the top cover.
  • the system further comprises a ventilation tube.
  • the system further comprises a vacuum source.
  • the vacuum source comprises a centralized vacuum system.
  • the top plate is configured for attachment to the ventilation tube.
  • the ventilation tube is configured for attachment to the vacuum source.
  • the present invention provides methods comprising forming a ventilation opening in a top plate of a top enclosure such that the top plate is configured for attachment to a ventilation tube.
  • the present invention provides methods comprising; a) providing; i) a top enclosure comprising a top plate, and ii) a ventilation tube; and b) forming a ventilation opening in the top plate, and c) attaching the ventilation tube to the top plate such that the ventilation tube forms a seal around the ventilation opening.
  • the methods further comprise step d) attaching a least one panel to the top enclosure.
  • the present invention provides methods comprising; a) providing; i) a top cover of a nucleic acid synthesizer comprising a ventilation slot, wherein the top cover is configured to be attached to a top surface of a nucleic acid synthesizer such that the top surface may be raised and lowered; and ii) a top enclosure, wherein the top enclosure comprises a top plate with a ventilation opening, and b) attaching the top enclosure to the top cover such that a primarily enclosed space is formed over the top cover.
  • the methods further comprise the step of attaching at least one panel to the top enclosure (or the top cover), wherein the at least one panel extends at least part way between at least one side of the top cover (or the top cover) and the top surface such that at least a partial barrier to air is created on at least one side of the synthesizer when the top cover is extended upward such that it is not in contact with the top surface.
  • the present invention provides methods comprising; a) providing; i) a nucleic acid synthesizer comprising; i) a top cover with a ventilation slot, and ii) a top enclosure, wherein the top enclosure comprises a top plate with a ventilation opening, wherein the top enclosure is attached to the top cover to form a primarily enclosed space above the top cover, and wherein the top plate is attached to a ventilation tube such that the ventilation tube forms a seal around the ventilation opening, and ii) a vacuum source attached to the ventilation tube, and b) activating the vacuum source such that air is drawn into the ventilation slot, through the primarily open space, and out through the ventilation opening into the ventilation tube.
  • kits comprising; a) a top enclosure comprising a top plate with a ventilation opening, wherein the top enclosure is configured for attachment to a top cover of a synthesizer to form a primarily enclosed space over the top cover, and b) a printed material component, wherein the printed material component comprises written instruction for installing the top enclosure onto the top cover.
  • kits comprising; a) a panel configured to extend at least part way between at least one side of a top cover (or lid enclosure) and a top surface of a nucleic acid synthesizer such that at least a partial barrier to air is created on at least one side of the synthesizer when the top cover is extended upward such that it is not in contact with the top surface, and b) a printed material component, wherein the printed material component comprises written instructions for installing the panel onto a top cover (or lid enclosure).
  • the present invention relates to polymer synthesizers and methods of using polymer synthesizers.
  • the present invention provides highly efficient, reliable, and safe synthesizers that find use, for example, in high throughput and automated nucleic acid synthesis.
  • the present invention also relates to synthesizer anays for efficient, safe, and automated processes for the production of large quantities of oligonucleotides.
  • the present invention provides a system comprising a closed system solid phase synthesizer configured for parallel synthesis (e.g., simultaneous side-by-side synthesis) of three or more polymers (e.g., 3, 4, 5, 6, 1, . . ., 10, . . ., 48, . . ., 96, . . . ).
  • the present invention is not limited by the nature of the polymer.
  • Polymers include, but are not limited to, nucleic acids and polypeptides.
  • the nucleic acid polymers comprise DNA.
  • the DNA comprises an oligonucleotide.
  • the synthesizers of the present invention allow parallel synthesis of multiple polymers.
  • Each of the synthesized polymers may be identical to one another (e.g., in composition, sequence, length, etc.) or may be different than one another (e.g., in composition, sequence, length, etc.).
  • the synthesizers of the present invention may be configured to simultaneously produce three or more distinct polymers (e.g., oligonucleotides).
  • the synthesizers of the present invention allow parallel processing of polymers, large numbers of polymers may be produced in a single synthesizer in a short period of time.
  • the synthesizer may be configured to produce 100 or more polymers per day.
  • the synthesizer may be configured to produce 1000-2000 or more polymers per day.
  • synthesizers may be configured to produce 2000 or more oligonucleotide per day (e.g., oligonucleotides containing 20-40 or more bases).
  • the produced polymers e.g. , 2000 or more produced polymers
  • the produced polymers are produced on a micro- scale, e.g., less than 5 nmole synthesis scale. In some prefened embodiments, micro-scale synthesis is performed on a 0.1 to 1 nmole synthesis scale.
  • the present invention also provides a solid phase synthesizer comprising: a reaction support comprising three or more (e.g., 3, 4, 5, 6, 1, . . ., 10, . . ., 48, . . ., 96, . . . ) reaction chambers (e.g., chambers that are isolated from one another, such that fluid does not pass from one chamber to another during synthesis); and a plurality of reagent dispensers configured to simultaneously form closed fluidic connections with each of the reaction chambers, wherein the reagent dispensers are each configured to deliver all reagents necessary for a polymer synthesis reaction.
  • the reaction chambers comprise synthesis columns.
  • the reaction support provides a fixed surface to support three or more synthesis columns.
  • the synthesis columns comprise nucleic acid synthesis columns (e.g., columns designed for use with EXPEDITE nucleic acid synthesizers [Applied Biosystems, Foster City, CA], 3900 High-Throughput Columns for use with the 3900 DNA Synthesizer [Applied Biosystems], DNA synthesis columns from Biosearch Technologies, Novato, CA).
  • the reaction support is configured to contain and form a tight seal around multiple, different synthesis columns (e.g., of different sizes or from different manufacturers), so as to allow any number of commercially available columns to be used with the synthesizer.
  • the reagent dispensers are fluidicly connected to a plurality of reagent tanks (e.g., through tubing).
  • reagent dispensers are constructed from any substantially inert materials including, but not limited to, stainless steel, glass, Teflon, and titanium.
  • Tanks include, but are not limited to, acetonitrile tanks, phosphoramidite tanks, argon gas tanks, oxidizer tanks, tetrazole tanks, and capping solution tanks.
  • the tanks are contained within the synthesizer. In other embodiments, the tanks are contained on an outer surface of the synthesizer.
  • tanks are provided separately from the synthesizer (e.g., in a different room, such as an explosion-proofroom).
  • the present invention provides large volume synthesis facilities containing multiple synthesizers, wherein two or more of the synthesizer are serviced by the same reagent tanks.
  • "large volume containers" are used as reagent tanks.
  • Individual large volume reagent tanks contain from about 200 liters to about 2500 liters of acetonitrile, from about 200 liters to about 2500 liters of deblocking solution; from about 2 liters to about 200 liters of amidite; from about 20 liters to about 200 liters of activator (e.g., tetrazol); from about 20 liters to about 200 liters of capping reagents; or from about 20 liters to about 200 liters of oxidizer.
  • a plurality of tanks containing a combined capacity as indicated above may be used.
  • the large volume reagent tanks are connected to a plurality of synthesizers through a large volume reagent delivery system, which allows large volumes of reagents to be delivered simultaneously to each of the synthesizers
  • the reaction support comprises a fixed reaction support (e.g., a reaction support that does not move during operation).
  • the reaction support comprises a plurality of waste channels. In prefened embodiments, the waste channels in closed fluidic contact with each of the reaction chambers (See e.g., Figure 53).
  • the synthesizer further comprises providing energy, such as heat to the reaction chambers.
  • Heating of the reaction chamber finds use, for example, in decreasing the coupling time during a nucleic acid synthesis. It can also broaden the range of the chemical protocols that can be used in high throughput synthesis, e.g. by improving the efficiency of less efficient chemistries, such as the phosphate triester method of oligonucleotide synthesis.
  • the synthesizer further comprises a mixing component, such as an agitator, configured to agitate the reaction chambers (e.g., to mix reaction components, and to facilitate mass exchange between the reaction medium and the solid support).
  • the present invention further provides a solid phase synthesizer comprising: a fixed reaction support comprising three or more reaction chambers; and a plurality of reagent dispensers configured to simultaneously form closed fluidic connections with each of said reaction chambers.
  • the present invention also provides integrated systems that link nucleic acid synthesizers to other nucleic acid production components.
  • the present invention provides a system comprising a closed system nucleic acid synthesizer and a cleavage and deprotect component.
  • the synthesizer is configured for parallel synthesis of nucleic acid molecules at three or more reaction sites.
  • the system further comprises a reaction support comprising three or more reaction chambers, wherein the reaction support is configured for operation with both the nucleic acid synthesizer and the cleavage and deprotect component.
  • the system further comprises sample tracking software configured to associate sample identification tags (e.g., electronic identification numbers, barcodes) with samples that are processed by the nucleic acid synthesizer and the cleavage and deprotect component.
  • sample identification tags e.g., electronic identification numbers, barcodes
  • the sample fracking software is further configured to receive synthesis request information from a user, prior to sample processing by the nucleic acid synthesizer.
  • the system further comprises a robotic component configured to transfer the reaction support from the nucleic acid synthesizer to the cleavage and deprotect component.
  • the robotic component is further configured to transfer the reaction support from the cleavage and deprotect component to a purification component and/or to additional production components described herein.
  • the present invention also provides control systems for operating one or more components of the systems of the present invention.
  • the present invention provides a system comprising a processor, wherein the processor is configured to operate a close system nucleic acid synthesizer for parallel synthesis of three or more nucleic acid molecules.
  • the present invention further provides a system comprising a processor, wherein said processor is configured to operate a nucleic synthesizer and a cleavage and deprotect component.
  • the system further comprises a computer memory, wherein the computer memory comprises nucleic acid sample order information (e.g., information obtained from a user specifying the identity of a polymer to be synthesized and/or specifying one or more characteristics of the polymer such as sequence information).
  • nucleic acid sample order information e.g., information obtained from a user specifying the identity of a polymer to be synthesized and/or specifying one or more characteristics of the polymer such as sequence information.
  • the computer memory further comprises allele frequency information and/or disease association information.
  • the present invention relates to detecting mutations in pooled nucleic acid samples.
  • the present invention relates to compositions and methods for detecting mutations or measuring allele frequencies in pooled nucleic acid samples employing the INVADER detection assay or other detection assays described herein.
  • the present invention provides methods for detecting an allele frequency of a polymo ⁇ hism, comprising: a) providing; i) a pooled sample, wherein the pooled sample comprises target nucleic acid sequences from at least 10 individuals (or at least 50, or at least 100, or at least 250, or at least 500, or at least 1000 individuals, etc.); and ii) INVADER detection reagents (e.g.
  • a primary probes INVADER oligonucleotides, FRET cassettes, a structure specific enzyme, etc.
  • INVADER detection reagents configured to detect the presence or absence of a polymo ⁇ hism
  • c) measuring the detectable signal thereby determining a number of the target nucleic acid sequences that contain the polymo ⁇ hism (e.g. a quantitative number of molecules, or the allele frequency for the polymo ⁇ hism in a population, is determined).
  • signals from two or more alleles for a particular target nucleic acid locus are measured and the numbers are compared.
  • the measurements for two or more different alleles of a particular target nucleic acid locus are measured in a single reaction. In other embodiments, measurements from one or more alleles of a particular target nucleic acid locus are compared to measurements from one or more reference target nucleic acid loci. In prefened embodiments, measurements from one or more alleles of a particular target nucleic acid locus are compared to measurements from one or more reference target nucleic acid loci in the same reaction mixture. Further methods allow a single individual's particular allele frequency (i.e., frequency of the mutation among multiple copies of the sequence within an individual) or quantitative number of molecules found to possess the polymo ⁇ hism (e.g.
  • the individuals are from the same racial or ethnic class (e.g. European, African, Asian, Mexican, etc).
  • the present invention provides methods for detecting a rare mutation comprising; a) providing; i) a sample from a single subject, wherein the sample comprises at least 10,000 target nucleic acid sequences (e.g. from 10,000 cells, or at least 20,000 target nucleic acid sequences, or at least 100,000 target nucleic acid sequences), ii) a detection assay (e.g. the INVADER assay) capable of detecting a mutation in a population of target nucleic acid sequence that is present at an allele frequency of 1 : 1000 or less compared to wild type alleles; and b) assaying the sample with the detection assay under conditions such that the presence or absence of a rare mutation (e.g.
  • the target nucleic acid sequences are genomic (e.g. not polymerase chain reaction, or PCR, amplified, but directly from a cell). In other embodiments, the target nucleic acid sequences are amplified (e.g., by PCR).
  • the present invention provides methods for detecting a rare mutation comprising; a) providing: i) a sample from a single subject, wherein the sample comprises at least 10,000 target nucleic acid sequences, ii) a detection assay capable of detecting a mutation in a population of target nucleic acid sequence that is present at an allele frequency of 1 : 1000 or less compared to wild type alleles; and b) assaying the sample with the detection assay under conditions such that an allele frequency in the sample of a rare mutation is determined.
  • the subject's allele frequency is compared statistically to a known reference allele frequency (e.g. determined by the methods of the present invention or other methods), such that a diagnosis may be made (e.g. extent of disease, likelihood of having the disease, or passing it on to offspring, etc).
  • the present invention also provides methods for determining the number of molecules of one or more polymo ⁇ hisms present in a sample by employing, for example, the INVADER assay (e.g. polymo ⁇ hisms such as SNPs that are associated with disease).
  • This assay may be used to determine the number of a particular polymp ⁇ hism in a first sample, and then determining if there is a statistically significant difference between that number and the number of the same polymo ⁇ hism in a second sample.
  • one sample represents the number of the polymo ⁇ hism expected to occur in a sample obtained from a healthy individual, or from a healthy population if pooled samples are used.
  • a statistically significant difference between the number of a polymo ⁇ hism expected to be at a single-base locus in a healthy individual and the number determined to be in a sample obtained from a patient is clinically indicative.
  • the present invention relates to detection assay panels comprising an array of different detection assays.
  • the detection assays include assays for detecting mutations in nucleic acid molecules and for detecting gene expression levels. Assays find use, for example, in the identification of the genetic basis of phenotypes, including medically relevant phenotypes and in the development of diagnostic products, including clinical diagnostic products.
  • the present invention also provides systems and methods for data storage, including data libraries and computer storage media comprising detection assay data.
  • the present invention provides a panel comprising an anay, wherein the anay comprises a plurality of different assays (e.g., greater than about 50 different assays).
  • the assays are substantially similar to at least one assay shown in figure 96.
  • the anays comprise greater than about 100 different assays (e.g., 100, 101, 102, . . ., 130, . . ., 500, . . ., 1000, . . ., 10,000, . . ., 30,000, . . .).
  • the assays comprise biplex assays.
  • the assays comprise multiplex assays.
  • the array is a microanay.
  • the assays are provided on a solid surface.
  • the assays are provided on a microtiter plate.
  • the assays comprise nucleic acid detection assay.
  • the assays detect polymo ⁇ hisms (e.g., single-nucleotide polymo ⁇ hisms in nucleic acids), including direct detection of genomic DNA (e.g., human genomic DNA).
  • the present invention also provides methods for using panels.
  • the present invention provides a method comprising: a) providing: i) a panel comprising an array, said anay comprising a plurality of different assays (e.g., detection assays) and ii) a sample; and b) exposing the sample to the panel under conditions such that at least one of the assays detects the presence of a target nucleic acid in the sample. Any of the panels or detection assays described herein may be used in the method..
  • the present invention also provides system and methods for developing clinical products based on information obtained from the use of the panels.
  • Systems and methods are also provided for collecting, storing, and analyzing information obtained from use of the panels.
  • the present invention provides data libraries comprising data collected from detection assay testing.
  • the data libraries contain data obtained from an assay similar to at least one assay shown in Figure 96.
  • the data libraries contain information obtained from greater than about 100 different assays (e.g., 100, 101, 102, . . ., 130, . . ., 500, . . .).
  • data libraries include test result data including, but not limited to, the presence or absence of a mutation in nucleic acid from a sample, allele frequency information, quantitation data, and disease conelation data.
  • the data libraries also provide information conelated to the test result data including, but not limited to, an identity of a testing facility, detection assay components used to generate the data, other related detection assay components, reaction conditions, the identity of a user who requested the manufacture of the detection assay, date of detection assay use and/or testing, detection assay reliability information (e.g., determined the in silico methods of the present invention), information pertaining to the target sequence intenogated by the detection, information pertaining to clinical approval or requirements, and the like.
  • the present invention provides computer storage medium containing the above information and systems and methods for storing, accessing, and retrieving the information.
  • the present invention further provides methods for simultaneously detecting a plurality of polymo ⁇ hisms (e.g., SNPs).
  • a plurality of polymo ⁇ hisms e.g., SNPs
  • the present invention provides systems and methods for simultaneously detecting 100 or more polymo ⁇ hism (100, . . ., 1000, . . ., 10,000, . . ., 100,000, . . .).
  • the plurality of polymo ⁇ hisms are detected in a single reaction sample (e.g., in a multiplex reaction).
  • the polymo ⁇ hisms are present in genomic DNA and target sequences containing a single polymo ⁇ hism are amplified prior to detection of the polymo ⁇ hisms.
  • the amplification comprises PCR amplification. In some embodiments, amplification is carried out such that there is a 10 5 - 10 6 -fold increase in copies of the target sequence.
  • the present invention further provides system and methods for developing detection assays based on the design of a pre- validated detection assay. For example, the present invention provides thousands of specific INVADER detections assays directed at different target nucleic acid sequences, as well as components that find use in other detection assay formats. In some embodiments, one or more components of these assays are used in or are used in the design of a different type of detection assay. For example, validated target sequences may be used as targets in other types of detection assay.
  • oligonucleotides that hybridize to target sequences may be used directly, or in the design of hybridization oligonucleotides for other types of detection assays.
  • the present invention is not limited in the nature of the detection assay that is produced using information from the thousands of INVADER detection assays (e.g., assays described in Figure 96).
  • Such detection assays include, but are not limited to, hybridization methods and anay technologies (e.g., Aclara BioSciences, Haywood, CA; Affymetrix, Santa Clara, CA; Agilent Technologies, Inc., Palo Alto, CA; Aviva Biosciences Co ⁇ ., San Diego, CA; Caliper Technologies Co ⁇ ., Palo Alto, CA; Celera, Rockville, MD; CuraGen Co ⁇ ., New Haven, CT; Hyseq Inc., Sunnyvale, CA; Illumina, Inc., San Diego, CA; Incyte Genomics, Palo Alto, CA; Motorola BioChip Systems; Nanogen, San Diego, CA; Orchid BioSciences, Inc., Princeton, NJ; Applera Co ⁇ ., Foster City, CA; Rosetta Inpharmatics, Kirkland, WA; and Sequenom, San Diego, CA); polymerase chain reaction; branched hybridization methods; enzyme mismatch cleavage methods; NASB A; sandwich hybridization methods; methods employing molecular beacons
  • the present invention relates to systems and methods for managing genetic information and medical records.
  • the present invention provides systems and methods for collecting, storing, and retrieving patient-specific genetic information from one or more electronic databases.
  • the present invention provides an electronic medical record comprising genetic information of a subject (e.g., single nucleotide polymo ⁇ hism data of an animal or human patient) conelated to electronic medical history data of said subject.
  • a subject e.g., single nucleotide polymo ⁇ hism data of an animal or human patient
  • the present invention is not limited by the nature of the medical history data.
  • prescription data e.g., data related to one or more drugs or other prescribed medical interventions of the subject, including drug identity, drug reaction data, allergies, risk assessment data, and multi-drug interaction data, billing code levels, order restrictions
  • information pertaining a physician visit e.g., date and time of visit, identity of physicians, physician notes, diagnosis information, differential diagnosis information, patient location, patient status, order status, refenal information
  • patient identification information e.g., patient age, gender, race, insurance carrier, allergies, past medical history, family history, social history, religion, employer, guarantor, address, contact information, patient condition code
  • laboratory information e.g., labs, radiology, and tests.
  • the genetic information comprises single nucleotide polymo ⁇ hism data (e.g., data related to the presence of one or more single nucleotide polymo ⁇ hisms in the genetic material of the subject, including, but not limited to, the identity of the polymo ⁇ hisms, the location of the polymo ⁇ hisms, medical conditions associated with the presence or absence of the polymo ⁇ hisms, detection assays information) and/or information related to single nucleotide polymo ⁇ hism data (e.g., allele frequency of the polymo ⁇ hism in one or more populations).
  • single nucleotide polymo ⁇ hism data e.g., data related to the presence of one or more single nucleotide polymo ⁇ hisms in the genetic material of the subject, including, but not limited to, the identity of the polymo ⁇ hisms, the location of the polymo ⁇ hisms, medical conditions associated with the presence or absence of the polymo ⁇ hisms, detection assays
  • the single nucleotide polymo ⁇ hism data comprises data derived from an in vitro diagnostic single nucleotide polymo ⁇ hism detection assay. In some embodiments, the single nucleotide polymo ⁇ hism data comprises data derived from a panel comprising a plurality of single nucleotide polymo ⁇ hism detection assays. In some prefened embodiments, the panel comprises a detection assays that detects medically associated single nucleotide polymo ⁇ hisms (e.g., single nucleotide polymo ⁇ hisms associated with a disease).
  • the detection assays detect polymo ⁇ hisms associated with one or more medically relevant subject areas including, but not limited to cardiovascular disease, oncology, immunology, metabolic disorders, neurological disorders, musculoskeletal disorders, endocrinology, and genetic disease.
  • the panel comprises a plurality of single nucleotide polymo ⁇ hism detection assays associated with two or more diseases.
  • the panel comprises a plurality of single nucleotide polymo ⁇ hism detection assays that detect polymo ⁇ hisms in drug metabolizing enzymes.
  • the single nucleotide polymo ⁇ hism data comprises data derived from a plurality of in vitro diagnostic single nucleotide polymo ⁇ hism detection assays.
  • the detection assays comprises two or more unique invasive cleavage assays (INVADER assay, Third Wave Technologies, Madison, WI).
  • one or more of the two or more unique invasive cleavage assays detected at least one single nucleotide polymo ⁇ hism.
  • the single nucleotide polymo ⁇ hism is associated with a medical condition.
  • the two or more unique invasive cleavage assays comprise at least 10 unique detection assays (e.g., 10, 11, 12, . . ., 100, . . ., 1000, . . ., 10,000, . . ., 50,000, . . . ).
  • the single nucleotide polymo ⁇ hism data is derived from an analyte-specific reagent assay. In some embodiments, the single nucleotide polymo ⁇ hism data is derived from at least one clinically valid detection assay.
  • the electronic medical records of the present invention may be located on any number of computers or devices. For example, in some embodiments, the electronic medical record is contained in a computer system of a patient, an insurance company, a health care provider (e.g., a physician, a hospital, a clinic, a health maintenance organization), a government agency, and a drug retailer or drug wholesaler, or pharmaceutical company.
  • the electronic medical record is stored on a small device to be carried on or in a subject (e.g., a personal digital assistant, a MED-ALERT bracelet, a smart card, and an implanted data storage device such as those described in U.S. Pat. No. 5,499,626, herein inco ⁇ orated by reference in its entirety).
  • a small device to be carried on or in a subject
  • a subject e.g., a personal digital assistant, a MED-ALERT bracelet, a smart card, and an implanted data storage device such as those described in U.S. Pat. No. 5,499,626, herein inco ⁇ orated by reference in its entirety.
  • the electronic medical record comprises addition information, including, but not limited to, medical billing data, insurance claim data, and scheduling data.
  • the present invention also provides a computer system comprising the electronic medical records described herein.
  • the computer system is configured for receiving data from the Internet (e.g., e.g., single nucleotide polymo ⁇ hism data or one or more SNP assay(s) result data).
  • the computer system comprises one or more hardware or software components configured to carry out a processing routine.
  • a software application is configured to receive single nucleotide polymo ⁇ hism data automatically via a communications network.
  • the computer system comprises a routine for categorizing data (e.g., by disease type, by patient type, by genetic loci, etc.).
  • the computer system comprises a routine for carrying out a bioinformatics analysis routine (e.g., as described elsewhere herein).
  • the computer system comprises a routine for carrying out a mathematical manipulation routine.
  • the present invention further provides a method for determining a conelation between a polymo ⁇ hism (e.g., a SNP) and a phenotype, comprising: a) providing: samples from a plurality of subjects; medical records from the plurality of subjects, wherein the medical records contain information pertaining to a phenotype of the subjects; and detection assays that detect a polymo ⁇ hism; b) exposing the samples to the detection assays under conditions such that the presence or absence of at least one polymo ⁇ hism is revealed; and; c) determining a conelation between the at least one polymo ⁇ hism and the phenotype of the subjects.
  • a polymo ⁇ hism e.g., a SNP
  • the plurality of subjects comprises 1000 or more subjects (e.g., 10,000 or more subjects).
  • the information pertaining to a phenotype comprises information pertaining to a disease.
  • the information pertaining to a phenotype comprises information pertaining to a drug interaction.
  • the medical record comprises an electronic medical record. While the present invention is not limited by the nature of the sample, in some prefened embodiments, the sample comprises a blood sample or a tissue biopsy.
  • the present invention also provides an electronic library comprising a plurality of electronic medical records for different subjects, each of the electronic medical records comprising, polymo ⁇ hism data (e.g., single nucleotide polymo ⁇ hism data) of the subject conelated to electronic medical history data of the subject.
  • the electronic medical history data comprises prescription data.
  • the prescription data comprises drug reaction data.
  • the single nucleotide polymo ⁇ hism data comprises data derived from one or more in vitro diagnostic single nucleotide polymo ⁇ hisms detection assays.
  • the single nucleotide polymo ⁇ hism data comprises data derived from a panel, said panel comprising a plurality of single nucleotide polymo ⁇ hisms detection assays.
  • the panel comprises detection assays that detect medically associated single nucleotide polymo ⁇ hisms.
  • the panel comprises a plurality of single nucleotide polymo ⁇ hisms detection assays that detect single nucleotide polymo ⁇ hisms associated with a disease.
  • the panel comprises a plurality of detection assays that detect polymo ⁇ hisms associated with one or more medically relevant subject areas including, but not limited to, cardiovascular disease, oncology, immunology, metabolic disorders, neurological disorders, musculoskeletal disorders, endocrinology, and genetic disease.
  • the panel comprises a plurality of single nucleotide polymo ⁇ hism detection assays associated with two or more diseases.
  • the panel comprises a plurality of single nucleotide polymo ⁇ hism detection assays that detect polymo ⁇ hisms in drug metabolizing enzymes.
  • the single nucleotide polymo ⁇ hism data comprises data derived from a plurality of in vitro diagnostic single nucleotide polymo ⁇ hism detection assays for each said different subject.
  • the detection assays comprises two or more unique invasive cleavage assays.
  • the one or more of the two or more unique invasive cleavage assays detected at least one single nucleotide polymo ⁇ hism.
  • the at least one single nucleotide polymo ⁇ hism is associated with a medical condition.
  • the present invention is not limited by the number of unique invasive cleavage assays used in the method.
  • the two or more unique invasive cleavage assays comprise at least 10 unique detection assays (e.g., at least 1000, 10,000, 35,000, or more).
  • the single nucleotide polymo ⁇ hism data for each of the different subjects is derived from an analyte-specific reagent assay. In some embodiments, the single nucleotide polymo ⁇ hism data for each of the different subjects is derived from at least one clinically valid detection assay.
  • the present invention also provides computer systems comprising the electronic libraries.
  • the computer system is configured for securely receiving single nucleotide polymo ⁇ hism data from the Internet.
  • the computer system further comprises a routine to receive single nucleotide polymo ⁇ hism data for each of the different subjects automatically via a communications network.
  • the computer system further comprises a routine to receive single nucleotide polymo ⁇ hism data for each the different subjects from nodes of a national, regional or world-wide communications network.
  • the computer system further comprises a software application for categorizing the data for the different subjects.
  • the computer system further comprises a software application for carrying out a bioinformatics analysis on said data for each said different subject.
  • the present invention provides systems and methods for acquiring and analyzing biological information.
  • the present invention provides systems and methods for developing detection assays and for use of detection assays in basic research discovery to facilitate selection and development of clinical detection assays.
  • the present invention provides methods of validating a detection assay, comprising: a) collecting test result data from a plurality of users, wherein the test result data is generated with one or more detection panels, and wherein the detection panels comprise a plurality of candidate detection assays configured for target detection; and b) processing at least a portion of the test result data such that at least one valid detection assay is identified from the plurality of candidate detection assays.
  • the method further comprises step c) marketing said valid detection assay as an Analyte-Specific Reagent or an In- Vitro Diagnostic.
  • said marketing comprises selling and/or advertising.
  • the present invention provides methods of validating a detection assay, comprising: a) distributing one or more detection panels to a plurality of users, wherein the detection panels comprise a plurality of candidate detection assays configured for target detection; b) collecting test result data from at least a portion of the plurality of users, wherein the test result data is generated with the detection panels; and c) processing at least a portion of the test result data such that at least one valid detection assay is identified from the plurality of candidate detection assays.
  • the method further comprises step d) marketing said valid detection assay as an Analyte-Specific Reagent or an In- Vitro Diagnostic.
  • said marketing comprises selling and/or advertising.
  • the plurality of detection assays comprise two or more unique detection assays (e.g. 10, ... 50, .... 100, ... 1000, or more unique detection assays). In some embodiments, the plurality of detection assays comprise two or more unique INVADER assays (e.g. 10, ... 50, .... 100, ... 1000, or more unique INVADER assays).
  • the methods of the present invention further comprise a distribution system, wherein the distributing is accomplished with the distribution system.
  • the distributing one or more detection panels to the plurality of users is at a reduced cost.
  • the distributing one or more detection panels to the plurality of users is at a subsidized cost.
  • the distributing one or more detection panels to the plurality of users is at no cost.
  • the method further comprises the step of employing one or more of the plurality of candidate detection assays to discover at least one single nucleotide polymo ⁇ hism.
  • the plurality of detection assays comprise INVADER assays.
  • the method prior to step a), the method further comprises the step of utilizing one or more of the plurality of candidate detection assays to associate a single nucleotide polymo ⁇ hism with a medical condition.
  • the plurality of detection assays comprise INVADER assay components.
  • the method further comprises the step of utilizing one or more of the plurality of candidate detection assays, and computer aided analysis, to associate a single nucleotide polymo ⁇ hism with a medical condition.
  • the plurality of detection assays comprise INVADER assay components.
  • the INVADER assay components comprise an INVADER oligonucleotide, a probe, and a control target sequence.
  • the plurality of detection assays comprise TAQMAN assay components (e.g. a probe and control target sequence).
  • the one or more detection panel ' s are configured for detecting a marker associated with a disease category.
  • the disease category is selected from cardiovascular disease, cancer, autoimmune disease, metabolic disorders, neurological disease, musculoskeletal disorders, and endocrine related diseases.
  • the plurality of users comprise researchers. In other embodiments, the plurality of users comprises at least 10 individual users. In some embodiments, the plurality of users comprises at least 200 individual users. In particular embodiments, the plurality of users comprises at least 500 individual users. In still other embodiments, the plurality of users comprises at least 1000 individual users. In particular embodiments, the plurality of users comprises at least 10,000 individual users.
  • the plurality of detection assays comprises at least 10 unique detection assays. In other embodiments, the plurality of detection assays comprises at least 1000 unique detection assays. In particular embodiments, the plurality of detection assays comprises at least 10,000 unique detection assays. In certain embodiments, the plurality of detection assays comprises at least 50,000 unique detection assays. In particular embodiments, the method further comprises a step, after the processing step, of selling the at least one valid detection assay as an Analyte Specific Reagent (ASR).
  • ASR Analyte Specific Reagent
  • the method further comprises a step, after the processing step, of selling the at least one valid detection assay as an Analyte Specific Reagent (ASR) to an In- Vitro Diagnostic Manufacturer or to a non-clinical laboratory.
  • the method further comprises a step, after the processing step, of selling the at least one valid detection assay as an In- Vitro Diagnostic.
  • the test result data comprises raw assay data.
  • test result data comprises analyzed assay data.
  • the test result data comprises both raw assay data and analyzed assay data.
  • the test result data comprises data resulting from testing of at least separate samples (e.g. at least 1000, at least 10,000, or at least 100,000 separate samples).
  • the collecting comprises receiving the test result data from at least a portion of the plurality of users over a communications network (e.g. Internet or World Wide Web).
  • the collecting further comprises storing the test result data in a database.
  • the database is part of a computer system of a service provider.
  • the collecting comprises receiving the test result data over the Internet.
  • the collecting comprises retrieving the test result data from a user's computer system over a communication network.
  • the user's computer system comprises a software application configured to receive the test result data.
  • the software application is further configured to transmit the test result data automatically via a communications network.
  • the processing comprises categorizing the test result data (e.g. ananging the data according to unique detection assay and or type of medical condition associated with detection of a target). In other embodiments, the processing comprises in silico analysis. In certain embodiments, the processing comprises computer aided analysis of the test result data. In additional embodiments, the processing comprises mathematical manipulation of the test result data. In further embodiments, the processing comprises comparing the test result data to a substantially equivalent predicate assay. In particular embodiments, the processing comprises mathematical manipulation of the test result data, and comparing the test result data to a substantially equivalent predicate assay.
  • At least one valid detection assay is identified as a result of being substantially equivalent to a predicate assay, In some embodiments, processing at least a portion of the test result data generates assay validation information.
  • the methods of the present invention further comprise step e) submitting the assay validation information to a government body charged with approving products for clinical use.
  • the government body is the Food and Drug Administration.
  • the assay validation information is part of a 510(k) application that is submitted to the Food and Drug Administration.
  • the methods of the present invention further comprise a step of receiving approval from the Food and Drug Administration to market the at least one valid detection assay as an FDA approved In- Vitro diagnostic assay.
  • the FDA approved In- Vitro diagnostic assay is a predicate for determining substantially equivalency for other In- Vitro diagnostic assays.
  • the target is a single nucleotide polymo ⁇ hism (e.g. in a DNA or RNA molecule).
  • the target is RNA (e.g. such that RNA expression can be quantitated).
  • the present invention also provides a method of developing an in- vitro diagnostic DNA or RNA analysis product comprising, running an assay through a product development funnel, in which the assay that enters the product development funnel is substantially similar to the in- vitro diagnostic DNA or RNA analysis product.
  • the assay is an assay to detect a single nucleotide polymo ⁇ hism.
  • the product development funnel optionally comprises one or more of the following: a discovery portion, a medically associated portion, an analyte-specific reagent portion, and an in- vitro diagnostic portion.
  • the assay comprises a chromosome specific assay.
  • the method further comprises the step of using a panel, wherein the panel comprises the assay. In other embodiments, the panel comprises a whole genome panel.
  • the medically associated portion of the funnel comprises a panel organized by disease.
  • the panel organized by disease is selected from the group consisting of a cardiovascular disease panel, an oncology panel, an immunology panel, a metabolic disorders panel, a neurological disorders panel, a musculoskeletal disorders panel, an endocrinology panel, and a genetic disease panel.
  • the method further comprises the step of using a panel, wherein the panel is a panel for a multiplicity of disease states and/or wherein the panel comprises a drug metabolizing enzyme panel.
  • the present invention further provides a method of increasing revenue and/or a profit margin from the development of an in vitro diagnostic DNA or RNA analysis product comprising channeling an assay through a product development funnel, in which the assay is substantially similar to the in vitro diagnostic DNA or RNA analysis product.
  • the in vitro DNA or RNA analysis product comprises an FDA approved product.
  • the product development funnel has an ingress and an egress, wherein the assay is one of at least several thousand assays which enter the ingress. In other embodiments, the assay is one of about several hundred assays that exit the egress as the in vitro diagnostic DNA or RNA analysis product.
  • the present invention further provides a method of identifying single nucleotide polymo ⁇ hisms comprising providing: 1) a plurality of samples comprising genomic DNA from a first individual and four or more additional individuals, each of the first and four or more additional individuals having genomic DNA comprising a first region, said first individual having a first single nucleotide polymo ⁇ hism in the first region, 2) at least one detection reagent capable of generating a signal; and 3) at least one oligonucleotide .
  • the probe designed to cause the detection reagent to generate a signal following contact of the probe with a portion of the first region of the genomic DNA of the first individual; contacting each of the genomic DNA samples with the oligonucleotide probe under conditions such that a signal is detected for the genomic DNA of the first individual; identifying at least one of the four or more additional individuals for which no signal is detected, thereby identifying a negative-tested individual; and assaying the first region of the negative-tested individual under conditions such that a second single nucleotide polymo ⁇ hism is revealed in the first region of the genomic DNA of the negative- tested individual in addition to the first single nucleotide polymo ⁇ hism, wherein the first individual lacks the second single nucleotide polymo ⁇ hism.
  • the method further provides a second oligonucleotide probe designed to cause the detection reagent to generate a signal following contact of the probe with a portion of the first region of the genomic DNA of the negative-tested individual, wherein the second oligonucleotide probes is contacted with the genomic DNA sample of the negative-tested individual.
  • the second probe may be used concunently with the first probe or may be used after the first probe (e.g., experiments conducted with the first probe may lead to the design of a second probe e.g., using the systems and methods of the present invention).
  • the method may also include identifying negative detection assay results that are the result of one or more individuals lacking the first single nucleotide polymo ⁇ hism.
  • Figure 1 shows a general overview of the systems of the present invention.
  • Figured 2a-2f show various embodiments of INVADER LOCATOR computer interface displays.
  • Figure 3 shows an overview of in silico analysis in some embodiments of the present invention.
  • Figure 4 shows an overview of information flow for the design and production of detection assays in some embodiments of the present invention.
  • Figure 5 shows how the in silico processes of the present invention allow information to be processed to generate useful detection panels.
  • Figure 6 shows one embodiment of the INVADER detection assay.
  • Figure 7 shows a computer display of an INVADERCREATOR Order Entry screen.
  • Figure 8 shows a computer display of an INVADERCREATOR Multiple SNP Design Selection screen.
  • Figure 9 shows a computer display of an INVADERCREATOR Designer Worksheet screen.
  • Figure 10 shows a computer display of an INVADERCREATOR Output Page screen.
  • Figure 11 shows a computer display of an INVADERCREATOR Printer Ready Output screen.
  • Figure 12 A-12R show various SNP INVADER CREATOR (SIC) computer interface displays.
  • Figures 13A-13Q show various RIC INVADERCREATOR computer interface displays.
  • Figures 14a-14f show various TIC INVADER CREATOR computer interface displays.
  • Figure 15 shows an input target sequence and the result of processing this sequence with systems and routines of the present invention
  • Figure 16 shows an example of a basic work flow for highly multiplexed PCR using the
  • Figure 17 shows a flow chart outlining the steps that may be performed in order to generate a primer set useful in multiplex PCR.
  • Figures 18-22 show sequences used and data generated in connection with PCR Primer Design Example 1.
  • FIGS 23-30 show sequences used and data generated in connection with Example 2.
  • Figure 31 shows certain PCR primers useful for amplifying various regions of CYP2D6.
  • Figure 32 shows one protocol for Multiplex PCR optimization according to the present invention.
  • Figure 33 illustrates a perspective view of an exemplary synthesizer.
  • Figure 34 illustrates a cross-sectional view of an exemplary synthesizer.
  • Figure 35 illustrates a perspective view of a cartridge, chamber bowl and chamber seal of the present invention.
  • Figure 36 illustrates a detailed view of an exemplary cartridge.
  • Figure 37 illustrates an exemplary drain plate.
  • Figure 38A illustrates a top view of one embodiment of a drain plate.
  • Figure 38B illustrates a top view of another embodiment of a drain plate gasket.
  • Figure 39 illustrates a side view of a drain plate gasket situated between a cartridge and a drain plate.
  • Figure 40 illustrates a cross-sectional view of a waste tube system.
  • Figure 41 illustrates a chamber bowl with chamber drain.
  • Figures 42A-C illustrate different embodiments of energy input components 95 and mixing components 96.
  • Figures 43A-B illustrate different combinations of energy input components 95 and mixing components 96.
  • Figure 44 illustrates one embodiment of a synthesis column.
  • Figure 45 illustrates a computer system coupled to a synthesizer.
  • Figures 46A-C illustrate 3 cross-sectional detailed views of different embodiments of a cartridge, drain plate, drain plate gasket, receiving hole of cartridge, and synthesis column.
  • Figure 47A and 47B illustrate embodiments of reagent dispense stations.
  • Figure 48A illustrates a synthesizer having a ventilation opening in a lid enclosure.
  • Figures 48B and 48C illustrate a synthesizer having ventilation tubing attached to a ventilation opening in a lid enclosure.
  • Figures 49A-C illustrate synthesizers having ventilated workspaces.
  • Figures 50A and 50B provide cross sectional views of an exemplary synthesizer having a lid enclosure 102, and illustrate air flow 109 toward the ventilation tubing 103 when the lid enclosure 102 is in a closed or opened position, respectively.
  • Figures 51 A and 5 IB provide cross sectional views of an exemplary synthesizer having a primarily enclosed space in a base 2, and illustrate air flow 109 toward the ventilation tubing 103 when the lid enclosure 102 is in a closed or opened position, respectively.
  • Figure 52 illustrates a synthesizer 1, a robotic means 92, a cleave and deprotect component 93 and a purification component 94.
  • Figure 53 shows a schematic diagram of a polymer synthesizer of the present invention.
  • Figure 54A shows a side view of a reagent dispenser (2).
  • Figure 54B shows a cross- sectional view of a reagent dispenser (2).
  • Figures 55 A and 55B show a prefened embodiment of the reagent dispenser (2), wherein the outer surface of the delivery channel (9) contains first (13) and second (14) ring seals configured to form an airtight or substantially airtight seal with one or more points on the interior surface of a synthesis column (15) or other reaction chamber (e.g., with reaction chambers present in a synthesizer or a cleavage and deprotection component).
  • Figure 56 shows a solvent delivery component in one embodiment of the present, invention.
  • Figure 57 shows a waste storage and purge component in one embodiment of the present invention.
  • Figured 58A-K show flow charts depicting the integrated data and process flows employed in the oligonucleotide production systems of the present invention.
  • Figure 59A-D show various protocols for high throughput, automated genotyping.
  • Figure 60A-60H various embodiments of the cleave and deprotect devices, and components thereof, of the present invention.
  • Figure 61 shows one embodiment of a data management system of the present invention.
  • Figure 62 shows another embodiment of a data management system of the present invention.
  • Figure 63 shows a computer display of an association database.
  • Figure 64 shows a computer display of a Microsoft Excel worksheet having data received by export from an association database.
  • Figure 65 shows a computer display of a plate viewer.
  • Figure 66 shows a computer display of a data viewer.
  • Figure 67 shows a computer display of allele caller results, having SNP results data displayed in the cells.
  • Figure 68 shows a computer display of allele caller results, having analyzed input assay data (in this example, a calculated ratio) displayed in the cells.
  • Figure 69 shows a computer display of a Microsoft Excel worksheet having SNP results data received by export from an allele caller.
  • Figure 70 shows a graph demonstrating the ability of the INVADER assay to detect mutations in the APOC4 gene in pooled samples.
  • Figure 71 shows a graph demonstrating the ability of the INVADER assay to detect mutations in the CFTR gene in pooled samples.
  • Figures 72-75 show graphs of the results of experiments described in Pooled Sample - Example 3.
  • Figure 76A shows data measuring allele signals in INVADER assays for detection of alleles comprising the indicated percentages of the number of copies of each locus.
  • Figure 76B shows an Excel graph comparing theoretical allele frequencies to allele frequencies calculated from the INVADER assay data shown in Figure 5A.
  • Figure 77 shows an Excel graph and data comparing actual and calculated allele frequencies for each of 8 SNP loci detected in pooled genomic DNA from 8 different individuals.
  • Figure 78 shows an Excel graph and data showing calculated allele frequencies compared to fold-over-zero minus 1 (FOZ-1) measurements for SNP locus 132505 in genomic DNAs having different mixtures of these alleles.
  • Figure 79 shows an Excel graph and data showing calculated allele frequencies compared to fold-over-zero minus 1 (FOZ-1) measurements for SNP locus 131534 in genomic DNAs having different mixtures of these alleles.
  • Figures 80A-80C show the sequences of the probes configured for use in the assays described in Pooled Sample - Example 4 and synthetic targets for each allele.
  • "Y" indicates an amine blocking group. The polymo ⁇ hism and the dye that will be detected for each probe, when used in the exemplary assay configurations described in Example 4, are indicated.
  • Figure 81 shows an overview of the integration of components of the systems and methods of the present invention.
  • Figure 82 shows identified p4502D6 polymo ⁇ hisms.
  • Figure 83 shows CYP2D6 specific PCR amplification.
  • Figure 84 depicts biplex signal detection using INVADER assays to detect CYP2D6.
  • Figures 85 and 86 show the results of an INVADER assay screen of 175 individuals for various CYP2D6 polymo ⁇ hisms.
  • Figure 87 shows the minor allele frequency by population for various SNP consortium/Third Wave Technologies SNPs.
  • Figure 88 shows a schematic summary of the flow of detection assay development in the present invention from research products to clinical products.
  • Figure 89 shows a schematic summary of the discovery phase of the diagram shown in Figure 88.
  • Figure 90 shows a schematic summary of the development of potential clinical markers phase of the diagram shown in Figure 88.
  • Figure 91 shows exemplary detection assay products from each phase of the diagram shown in Figure 88.
  • Figure 92 shows business revenue generation from products from each phase of the diagram shown in Figure 88.
  • the arrows showing revenue/margin per detection assay are not quantitative, but simply show a qualitative increase for each layer of the funnel.
  • Figure 93 shows a flow chart depicting a disease associated assay development process.
  • Figure 94 shows an overview of an ASR Fast Track Process.
  • Figure 95 shows a flow chart depicting a process for identifying "Super SNPs.”
  • Figure 96 shows INVADER assay components for detecting polymo ⁇ hisms in certain genes.
  • Figure 97A-97D shows various steps in the quality control assessment methods and protocols of the present invention.
  • Figure 98 shows a general overview of the oligonucleotide production and processing systems of the present invention.
  • solid support refers to any material that provides a solid or semi-solid structure with which another material can be attached.
  • materials include smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces) as well as textured and porous materials.
  • Such materials also include, but are not limited to, gels, rubbers, polymers, and other non-rigid materials.
  • Solid supports need not be flat. Supports include any type of shape including spherical shapes (e.g., beads). Materials attached to solid support may be attached to any portion of the solid support (e.g., may be attached to an interior portion of a porous solid support material).
  • Prefened embodiments of the present invention have biological molecules such as nucleic acid molecules and proteins attached to solid supports.
  • a biological material is "attached" to a solid support when it is associated with the solid support through a non-random chemical or physical interaction. In some prefened embodiments, the attachment is through a covalent bond. However, attachments need not be covalent or permanent.
  • materials are attached to a solid support through a "spacer molecule" or “linker group.” Such spacer molecules are molecules that have a first portion that attaches to the biological material and a second portion that attaches to the solid support. Thus, when attached to the solid support, the spacer molecule separates the solid support and the biological materials, but is attached to both.
  • the term "derived from a different subject,” such as samples or nucleic acids derived from a different subjects refers to a samples derived from multiple different individuals.
  • a blood sample comprising genomic DNA from a first person and a blood sample comprising genomic DNA from a second person are considered blood samples and genomic DNA samples that are derived from different subjects.
  • a sample comprising five target nucleic acids derived from different subjects is a sample that includes at least five samples from five different individuals. However, the sample may further contain multiple samples from a given individual.
  • the term "treating together,” when used in reference to experiments or assays, refers to conducting experiments concunently or sequentially, wherein the results of the experiments are produced, collected, or analyzed together (i.e., during the same time period). For example, a plurality of different target sequences located in separate wells of a multiwell plate or in different portions of a microanay are treated together in a detection assay where detection reactions are carried out on the samples simultaneously or sequentially and where the data collected from the assays is analyzed together.
  • test result data refers to data collected from performance of an assay (e.g., to detect or quantitate a gene, SNP or an RNA).
  • Test result data may be in any form, i.e., it may be raw assay data or analyzed assay data (e.g., previously analyzed by a different process).
  • Collected data that has not been further processed or analyzed is refened to herein as "raw” assay data (e.g., a number conesponding to a measurement of signal, such as a fluorescence signal from a spot on a chip or a reaction vessel, or a number conesponding to measurement of a peak, such as peak height or area, as from, for example, a mass spectrometer, HPLC or capillary separation device), while assay data that has been processed through a further step or analysis (e.g., normalized, compared, or otherwise processed by a calculation) is refened to as "analyzed assay data” or "output assay data".
  • raw assay data e.g., a number conesponding to a measurement of signal, such as a fluorescence signal from a spot on a chip or a reaction vessel, or a number conesponding to measurement of a peak, such as peak height or area, as from, for example, a mass spectrometer, HPLC or ca
  • genomic information database refers to collections of information (e.g., data) arranged for ease of retrieval, for example, stored in a computer memory.
  • a "genomic information database” is a database comprising genomic information, including, but not limited to, polymo ⁇ hism information (i.e., information pertaining to genetic polymo ⁇ hisms), genome information (i.e., genomic information), linkage information (i.e., information pertaining to the physical location of a nucleic acid sequence with respect to another nucleic acid sequence, e.g., in a chromosome), and disease association information (i.e., information conelating the presence of or susceptibility to a disease to a physical trait of a subject, e.g., an allele of a subject).
  • Database information refers to information to be sent to databases, stored in a database, processed in a database, or retrieved from a database.
  • Sequence database information refers to database information pertaining to nucleic acid sequences.
  • sequence database information refers to database information pertaining to nucleic acid sequences.
  • distinct sequence databases refers to two or more databases that contain different information than one another. For example, the dbSNP and GenBank databases are distinct sequence databases because each contains information not found in the other.
  • centralized control system or “centralized control network” refer to information and equipment management systems (e.g., a computer processor and computer memory) operable linked to a module or modules of equipment (e.g., DNA synthesizers).
  • equipment management systems e.g., a computer processor and computer memory
  • module or modules of equipment e.g., DNA synthesizers
  • oligonucleotide synthesizer component refers to a component of a system that is capable of synthesizing oligonucleotides (e.g., a oligonucleotide synthesizers). In some embodiments, the oligonucleotide synthesizer component comprises a plurality of oligonucleotide synthesizers that are operably linked.
  • oligonucleotide processing component refers to a component of a system capable of processing of oligonucleotides post-synthesis.
  • oligonucleotide processing stations include, but are not limited to, purification stations, dry-down stations, cleavage and deprotection stations, desalting stations, dilute and fill stations, and quality control stations.
  • computer memory and “computer memory device” refer to any storage media readable by a computer processor. Examples of computer memory include, but are not limited to, RAM, ROM, computer chips, digital video disc (DVDs), compact discs (CDs), hard disk drives (HDD), and magnetic tape.
  • computer readable medium refers to any device or system for storing and providing information (e.g., data and instructions) to a computer processor. Examples of computer readable media include, but are not limited to, DVDs, CDs, hard disk drives, magnetic tape and servers for streaming media over networks.
  • processor and "central processing unit” or “CPU” are used interchangeably and refers to a device that is able to read a program from a computer memory * (e.g., ROM or other computer memory) and perfonn a set of steps according to the program.
  • a computer memory * e.g., ROM or other computer memory
  • oligonucleotide specification information refers to any information used during the production of an oligonucleotide.
  • oligonucleotide specification information includes, but is not limited to, sequence information, end-user (e.g., customer) information, and concentration information (e.g., the final concentration desired by the end-user).
  • the term "conesponding oligonucleotides” is used to refer to oligonucleotides that differ in at least one characteristic (e.g., sequence, purity, required buffer, required salt concentration) and that are to be provided together (e.g., in an INVADER assay, the INVADER oligonucleotide and Primary Probe are 'conesponding oligonucleotides').
  • the term "divergent production” refers to the production of conesponding oligonucleotides employing at least two manufacturing stations, where a first conesponding oligonucleotide is never processed by at least one manufacturing station that is used to process a conesponding oligonucleotide.
  • set of oligonucleotides means at least two oligonucleotides that differ in at least one characteristic (e.g., sequence, purity, required buffer, required salt concentration).
  • purified sample refers to a sample where the full-length oligonucleotide in a sample is the predominate species of oligonucleotide.
  • at least 90%, preferably 95%, and more preferably 99% of oligonucleotides in a sample are full-length oligonucleotides
  • SNP single nucleotide polymo ⁇ hisms
  • SNPs can be located in a portion of a genome that does not code for a gene.
  • a “SNP” may be located in the coding region of a gene. In this case, the "SNP” may alter the structure and function of the RNA or the protein with which it is associated.
  • allele refers to a variant form of a given sequence (e.g., including but not limited to, genes containing one or more SNPs).
  • a large number of genes are present in multiple allelic forms in a population.
  • a diploid organism carrying two different alleles of a gene is said to be heterozygous for that gene, whereas a homozygote carries two copies of the same allele.
  • the term “linkage” refers to the proximity of two or more markers (e.g., genes) on a chromosome.
  • allele frequency refers to the frequency of occunence of a given allele (e.g., a sequence containing a SNP) in given population (e.g., a specific gender, race, or ethnic group). Certain populations may contain a given allele within a higher percent of its members than other populations. For example, a particular mutation in the breast cancer gene called BRCAl was found to be present in one percent of the general Jewish population. In comparison, the percentage of people in the general U.S. population that have any mutation in
  • BRCAl has been estimated to be between 0.1 to 0.6 percent.
  • Two additional mutations, one in the BRCAl gene and one in another breast cancer gene called BRCA2 have a greater prevalence in the Ashkenazi Jewish population, bringing the overall risk for carrying one of these three mutations to 2.3 percent.
  • in silico analysis refers to analysis performed using computer processors and computer memory,
  • insilico SNP analysis refers to the analysis of
  • the term "genotype" refers to the actual genetic make-up of an organism
  • locus refers to the position of a gene or any other characterized sequence on a chromosome.
  • disease or “disease state” refers to a deviation from the condition regarded as normal or average for members of a species, and which is detrimental to an affected individual under conditions that are not inimical to the majority of individuals of that species (e.g., diarrhea, nausea, fever, pain, and inflammation etc).
  • treatment in reference to a medical course of action refers to steps or actions taken with respect to an affected individual as a consequence of a suspected, anticipated, or existing disease state, or wherein there is a risk or suspected risk of a disease state. Treatment may be provided in anticipation of or in response to a disease state or suspicion of a disease state, and may include, but is not limited to preventative, ameliorative, palliative or curative steps.
  • therapy refers to a particular course of treatment.
  • gene refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, RNA (e.g., rRNA, tRNA, etc.), or precursor.
  • RNA e.g., rRNA, tRNA, etc.
  • the polypeptide, RNA, or precursor can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., ligand binding, signal transduction, etc) of the full-length or fragment are retained.
  • the term also encompasses the coding region of a structural gene and the including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene conesponds to the length of the full-length mRNA.
  • the sequences that are located 5' of the coding region and which are present on the mRNA are refened to as 5' untranslated sequences.
  • the sequences that are located 3' or downstream of the coding region and that are present on the mRNA are refened to as 3' untranslated sequences.
  • gene encompasses both cDNA and genomic forms of a gene.
  • a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or
  • Introns are segments included when a gene is transcribed into heterogeneous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are generally absent in the messenger RNA (mRNA) transcript.
  • mRNA messenger RNA
  • Variations e.g., mutations, SNPS, insertions, deletions
  • transcribed portions of genes are reflected in, and can generally be detected in conesponding portions of the produced RNAs (e.g., hnRNAs, mRNAs, rRNAs, tRNAs).
  • amino acid sequence is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule
  • amino acid sequence and like terms, such as polypeptide or protein are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
  • genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences that are present on the RNA transcript. These sequences are refened to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript).
  • the 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene.
  • the 3' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage and polyadenylation.
  • wild-type refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source.
  • a wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal” or “wild-type” form of the gene.
  • the terms "modified,” “mutant,” and “variant” refer to a gene or gene product that displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
  • nucleic acid molecule encoding As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. In this case, the DNA sequence thus codes for the amino acid sequence.
  • DNA and RNA molecules are said to have "5' ends” and "3' ends” because mononucleotides are reacted to make oligonucleotides or polynucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage.
  • an end of an oligonucleotides or polynucleotide refened to as the "5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring.
  • a nucleic acid sequence even if internal to a larger oligonucleotide or polynucleotide, also may be said to have 5' and 3' ends.
  • an oligonucleotide having a nucleotide sequence encoding a gene and “polynucleotide having a nucleotide sequence encoding a gene,” means a nucleic acid sequence comprising the coding region of a gene or, in other words, the nucleic acid sequence that encodes a gene product.
  • the coding region may be present in either a cDNA, genomic DNA, or RNA form.
  • the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double-stranded.
  • Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and or conect processing of the primary RNA transcript.
  • the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, ete. or a combination of both endogenous and exogenous control elements.
  • the terms "complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules.
  • Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.
  • the term "homology” refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity).
  • a partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid and is refened to using the functional term "substantially homologous.”
  • the term “inhibition of binding,” when used in reference to nucleic acid binding, refers to inhibition of binding caused by competition of homologous sequences for binding to a target sequence. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency.
  • a substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
  • the absence of nonspecific binding may be tested by the use of a second target that lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
  • low stringency conditions factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions.
  • conditions that promote hybridization under conditions of high stringency e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
  • substantially homologous refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
  • a gene may produce multiple RNA species that are generated by differential splicing of the primary RNA transcript.
  • cDNAs that are splice variants of the same gene will contain regions of sequence identity or complete homology (representing the presence of the same exon or portion of the same exon on both cDNAs) and regions of complete non-identity (for example, representing the presence of exon "A” on cDNA 1 wherein cDNA 2 contains exon "B" instead). Because the two cDNAs contain regions of sequence identity they will both hybridize to a probe derived from the entire gene or portions of the gene containing sequences found on both cDNAs; the two splice variants are therefore substantially homologous to such a probe and to each other.
  • hybridization is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T m of the formed hybrid, and the G:C ratio within the nucleic acids.
  • T m is used in reference to the "melting temperature.”
  • the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • stringency is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. Those skilled in the art will recognize that “stringency” conditions may be altered by varying the parameters just described either individually or in concert. With “high stringency” conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences (e.g., hybridization under "high stringency” conditions may occur between homologs with about 85-100% identity, preferably about 70-100%) identity). With medium stringency conditions, nucleic acid base pairing will occur between nucleic acids with an intermediate frequency of complementary base sequences (e.g., hybridization under "medium stringency” conditions may occur between homologs with about 50-70% identity). Thus, conditions of
  • “weak” or “low” stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less.
  • “High stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42 C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO4 H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with
  • “Medium stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42 C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO4 H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO4 H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with
  • Low stringency conditions comprise conditions equivalent to binding or hybridization at 42 C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO4 H 2 O and 1.85 g/1
  • EDTA pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 g/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1 % SDS at 42 C when a probe of about 500 nucleotides in length is employed.
  • 5X Denhardt's reagent 50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 g/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1 % SDS at 42 C when a probe of about 500 nucleotides in length is employed.
  • reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA sequence given in a sequence listing or may comprise a complete gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.
  • two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window,” as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman [Smith and Waterman, Adv. Appl Math.
  • sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determimng the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the term "substantial identity” denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
  • the reference sequence may be a subset of a larger sequence, for example, as a splice variant of the full-length sequences.
  • the term "substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 99 percent sequence identity).
  • residue positions that are not identical differ by conservative amino acid substitutions.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic- hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Prefened conservative amino acids substitution groups are: valine- leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine- glutamine.
  • Amplification is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out.
  • Amplification enzymes are enzymes that, under conditions they are used, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid.
  • MDV-1 RNA is the specific template for the replicase (D.L. Kacian et al, Proc. Natl. Acad. Sci. USA 69:3038 [1972]).
  • Other nucleic acid will not be replicated by this amplification enzyme.
  • this amplification enzyme has a stringent specificity for its own promoters (M. Chamberlin et al, Nature 228:227 [1970]).
  • T4 DNA ligase the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (D.Y. Wu and R. B. Wallace, Genomics 4:560 [1989]).
  • Taq and Pfu polymerases by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non-target sequences (H.A. Erlich (ed.), PCR Technology, Stockton Press [1989]).
  • amplifiable nucleic acid is used in reference to nucleic acids that may be amplified by any amplification method. It is contemplated that "amplifiable nucleic acid” will usually comprise “sample template.”
  • sample template refers to nucleic acid originating from a sample that is analyzed for the presence of "target” (defined below).
  • background template is used in reference to nucleic acid other than sample template that may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.
  • the term "primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH).
  • the primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
  • the primer is an oligodeoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
  • probe or “hybridization probe” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, that is capable of hybridizing, at least in part, to another oligonucleotide of interest.
  • a probe may be single-stranded or doublestranded. Probes are useful in the detection, identification and isolation of particular sequences.
  • probes used in the present invention will be labeled with a "reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
  • target refers to a nucleic acid sequence or structure to be detected or characterized.
  • PCR polymerase chain reaction
  • K.B. Mullis See e.g, U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188, hereby inco ⁇ orated by reference
  • This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase.
  • the two primers are complementary to their respective strands of the double stranded target sequence.
  • the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule.
  • the primers are extended with a polymerase so as to form a new pair of complementary strands.
  • the steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle”; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence.
  • the length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter.
  • the method is refened to as the "polymerase chain reaction” (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified.”
  • PCR polymerase chain reaction
  • PCR With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; inco ⁇ oration of biotinylated primers followed by avidin-enzyme conjugate detection; inco ⁇ oration of 32p_i a beled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment).
  • any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules.
  • the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
  • PCR product refers to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.
  • amplification reagents refers to those reagents
  • telomere sequence DNA sequence, DNA sequence, etc.
  • amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).
  • amplification reagents refers to a DNA molecule that is comprised of segments of DNA joined together by means of molecular biological techniques.
  • antisense is used in reference to RNA sequences that are complementary to a specific RNA sequence (e.g., mRNA).
  • antisense strand is used in reference to a nucleic acid strand that is complementary to the "sense” strand.
  • the designation (-) i.e., “negative” is sometimes used in reference to the antisense strand, with the designation (+) sometimes used in reference to the sense (i.e., "positive") strand.
  • isolated when used in relation to a nucleic acid, as in “an isolated oligonucleotide” or “isolated polynucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA found in the state they exist in nature.
  • a given DNA sequence e.g., a gene
  • RNA sequences such as a specific mRNA sequence encoding a specific protein
  • isolated nucleic acids encoding a polypeptide include, by way of example, such nucleic acid in cells ordinarily expressing the polypeptide where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature.
  • the isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single-stranded or double-stranded form.
  • the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (t.e., the oligonucleotide or polynucleotide may single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).
  • portion when in reference to a nucleotide sequence (as in “a portion of a given nucleotide sequence”) refers to fragments of that sequence. The fragments may range in size from four nucleotides to the entire nucleotide sequence minus one nucleotide (e.g., 10 nucleotides, 11, . . ., 20, . . .).
  • purified or “to purify” refers to the removal of contaminants from a sample.
  • purified refers to molecules (e.g., nucleic or amino acid sequences) that are removed from their natural environment, isolated or separated.
  • isolated nucleic acid sequence is therefore a purified nucleic acid sequence.
  • substantially purified molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated.
  • recombinant protein or “recombinant polypeptide” as used herein refers to a protein molecule that is expressed from a recombinant DNA molecule.
  • native protein as used herein to indicate that a protein does not contain amino acid residues encoded by vector sequences; that is the native protein contains only those amino acids found in the protein as it occurs in nature.
  • a native protein may be produced by recombinant means or may be isolated from a naturally occurring source.
  • portion when in reference to a protein (as in “a portion of a given protein”) refers to fragments of that protein.
  • the fragments may range in size from four consecutive amino acid residues to the entire amino acid sequence minus one amino acid.
  • Southern blot refers to the analysis of DNA on agarose or acrylamide gels to fractionate the DNA according to size followed by transfer of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane.
  • the immobilized DNA is then probed with a labeled probe to detect DNA species complementary to the probe used.
  • the DNA may be cleaved with restriction enzymes prior to elecfrophoresis. Following elecfrophoresis, the DNA may be partially depurinated and denatured prior to or during transfer to the solid support.
  • Southern blots are a standard tool of molecular biologists (J. Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, pp 9.31-9.58 [1989]).
  • the term "Western blot” refers to the analysis of protein(s) (or polypeptides) immobilized onto a support such as nitrocellulose or a membrane.
  • the proteins are run on acrylamide gels to separate the proteins, followed by transfer of the protein from the gel to a solid support, such as nitrocellulose or a nylon membrane.
  • the immobilized proteins are then exposed to antibodies with reactivity against an antigen of interest.
  • the binding of the antibodies may be detected by various methods, including the use of labeled antibodies.
  • test compound refers to any chemical entity, pharmaceutical, drug, and the like that are tested in an assay (e.g., a drug screening assay) for any desired activity (e.g., including but not limited to, the ability to treat or prevent a disease, illness, sickness, or disorder of bodily function, or otherwise alter the physiological or cellular status of a sample).
  • Test compounds comprise both known and potential therapeutic compounds.
  • a test compound can be determined to be therapeutic by screening using the screening methods of the present invention.
  • a "known therapeutic compound” refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.
  • sample as used herein is used in its broadest sense.
  • a sample suspected of containing a human chromosome or sequences associated with a human chromosome may comprise a cell, chromosomes isolated from a cell (e.g., a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern blot analysis), RNA (in solution or bound to a solid support such as for Northern blot analysis), cDNA (in solution or bound to a sohd support) and the like.
  • a sample suspected of containing a protein may comprise a cell, a portion of a tissue, an extract containing one or more proteins and the like.
  • label refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect, and that can be attached to a nucleic acid or protein. Labels include but are not limited to dyes; radiolabels such as 32 P; binding moieties such as biotin; haptens such as digoxgenin; luminogenic, phosphorescent or fluorogenic moieties; and fluorescent dyes alone or in combination with moieties that can suppress or shift emission spectra by fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or abso ⁇ tion, magnetism, enzymatic activity, and the like.
  • a label may be a charged moiety (positive or negative charge) or alternatively, may be charge neutral.
  • Labels can include or consist of nucleic acid or protein sequence, so long as the sequence comprising the label is detectable.
  • signal refers to any detectable effect, such as would be caused or provided by a label or an assay reaction.
  • the term “detector” refers to a system or component of a system, e.g., an instrument (e.g. a camera, fluorimeter, charge-coupled device, scintillation counter, etc) or a reactive medium (X-ray or camera film, pH indicator, etc.), that can convey to a user or to another component of a system (e.g., a computer or controller) the presence of a signal or effect.
  • an instrument e.g. a camera, fluorimeter, charge-coupled device, scintillation counter, etc
  • a reactive medium X-ray or camera film, pH indicator, etc.
  • a detector can be a photometric or spectrophotometric system, which can detect ultraviolet, visible or infrared light, including fluorescence or chemiluminescence; a radiation detection system; a spectroscopic system such as nuclear magnetic resonance spectroscopy, mass spectrometry or surface enhanced Raman spectrometry; a system such as gel or capillary elecfrophoresis or gel exclusion chromatography; or other detection system known in the art, or combinations thereof.
  • a distribution system refers to systems capable of transferring and/or delivering materials from one entity to another or one location to another.
  • a distribution system for transferring detection panels from a manufacturer or distributor to a user may comprise, but is not limited to, a packaging department, a mail room, and a mail delivery system.
  • the distribution system may comprise, but is not limited to, one or more delivery vehicles and associated delivery personnel, a display stand, and a distribution center.
  • interested parties e.g., detection panel manufactures
  • the term "at a reduced cost” refers to the transfer of goods or services at a reduced direct cost to the recipient (e.g. user). In some embodiments, "at a reduced cost” refers to transfer of goods or services at no cost to the recipient.
  • the term "at a subsidized cost” refers to the transfer of goods or services, wherein at least a portion of the recipient's cost is defened or paid by another party. In some embodiments, "at a subsidized cost” refers to transfer of goods or services at no cost to the recipient.
  • the term "at no cost” refers to the transfer of goods or services with no direct financial expense to the recipient. For example, when detection panels are provided by a manufacturer or distributor to a user (e.g. research scientist) at no cost, the user does not directly pay for the tests.
  • detection refers to quantitatively or qualitatively identifying an analyte (e.g., DNA, RNA or a protein) within a sample.
  • detection assay refers to a kit, test, or procedure performed for the pu ⁇ ose of detecting an analyte nucleic acid within a sample.
  • Detection assays produce a detectable signal or effect when performed in the presence of the target analyte, and include but are not limited to assays inco ⁇ orating the processes of hybridization, nucleic acid cleavage (e.g., exo- or endonuclease), nucleic acid amplification, nucleotide sequencing, primer extension, or nucleic acid ligation.
  • nucleic acid cleavage e.g., exo- or endonuclease
  • nucleic acid amplification e.g., exo- or endonuclease
  • nucleotide sequencing e.g., primer extension, or nucleic acid ligation.
  • the term "functional detection oligonucleotide” refers to an oligonucleotide that is used as a component of a detection assay, wherein the detection assay is capable of successfully detecting (i.e., producing a detectable signal) an intended target nucleic acid when the functional detection oligonucleotide provides the oligonucleotide component of the detection assay. This is in contrast to a non-functional detection oligonucleotides, which fail to produce a detectable signal in a detection assay for the particular target nucleic acid when the non-functional detection oligonucleotide is provided as the oligonucleotide component of the detection assay. Determining if an oligonucleotide is a functional oligonucleotide can be carried out experimentally by testing the oligonucleotide in the presence of the particular target nucleic acid using the detection assay.
  • hyperlink refers to a navigational link from one document to another, or from one portion (or component) of a document to another.
  • a hyperlink is displayed as a highlighted word or phrase that can be selected by clicking on it using a mouse to jump to the associated document or documented portion.
  • hypertext system refers to a computer-based informational system in which documents (and possibly other types of data entities) are linked together via hyperlinks to form a user-navigable "web.”
  • Internet refers to any collection of networks using standard protocols.
  • the term includes a collection of interconnected (public and/or private) networks that are linked together by a set of standard protocols (such as TCP/IP, HTTP, and FTP) to form a global, distributed network. While this term is intended to refer to what is now commonly known as the Internet, it is also intended to encompass variations that may be made in the future, including changes and additions to existing standard protocols or integration with other media (e.g., television, radio, etc).
  • non-public networks such as private (e.g., co ⁇ orate) Intranets.
  • World Wide Web or “web” refer generally to both (i) a distributed collection of interlinked, user- viewable hypertext documents (commonly refened to as Web documents or Web pages) that are accessible via the Internet, and (ii) the client and server software components which provide user access to such documents using standardized Internet protocols.
  • the primary standard protocol for allowing applications to locate and acquire Web documents is HTTP, and the Web pages are encoded using HTML.
  • Web and World Wide Web are intended to encompass future markup languages and transport protocols that may be used in place of (or in addition to) HTML and HTTP.
  • the term "web site” refers to a computer system that serves informational content over a network using the standard protocols of the World Wide Web. Typically, a Web site conesponds to a particular Internet domain name and includes the content associated with a particular organization. As used herein, the term is generally intended to encompass both (i) the hardware/software server components that serve the informational content over the network, and (ii) the "back end” hardware/software components, including any non-standard or specialized components, that interact with the server components to perform services for Web site users.
  • HTML HyperText Markup Language
  • HTML is a standard coding convention and set of codes for attaching presentation and linking attributes to informational content within documents.
  • HTML is based on SGML, the Standard Generalized Markup Language.
  • HTML codes are embedded within the informational content of the document.
  • the Web document or HTML document
  • HTML tags can be used to create links to other Web documents (commonly refened to as "hyperlinks").
  • XML refers to Extensible Markup Language, an application profile that, like HTML, is based on SGML.
  • XML differs from HTML in that: information providers can define new tag and attribute names at will; document structures can be nested to any level of complexity; any XML document can contain an optional description of its grammar for use by applications that need to perform structural validation.
  • XML documents are made up of storage units called entities, which contain either parsed or unparsed data. Parsed data is made up of characters, some of which form character data, and some of which form markup. Markup encodes a description of the document's storage layout and logical structure.
  • XML provides a mechanism to impose constraints on the storage layout and logical structure, to define constraints on the logical structure and to support the use of predefined storage units.
  • a software module called an XML processor is used to read XML documents and provide access to their content and structure.
  • HTTP refers to HyperText Transport Protocol that is the standard World Wide Web client-server protocol used for the exchange of information (such as HTML documents, and client requests for such documents) between a browser and a Web server.
  • HTTP includes a number of different types of messages that can be sent from the client to the server to request different types of server actions. For example, a "GET" message, which has the format GET, causes the server to return the document or file located at the specified URL.
  • URL refers to Uniform Resource Locator that is a unique address that fully specifies the location of a file or other resource on the Internet.
  • the general format of a URL is protocol ://machine address :port/path/f ⁇ lename.
  • the port specification is optional, and if none is entered by the user, the browser defaults to the standard port for whatever service is specified as the protocol. For example, if HTTP is specified as the protocol, the browser will use the HTTP default port of 80.
  • PUSH technology refers to an information dissemination technology used to send data to users over a network.
  • World Wide Web a "pull” technology
  • PUSH protocols send the informational content to the user computer automatically, typically based on information pre-specified by the user.
  • a communication network refers to any network that allows information to be transmitted from one location to another.
  • a communication network for the transfer of information from one computer to another includes any public or private network that transfers information using electrical, optical, satellite transmission, and the like.
  • Two or more devices that are part of a communication network such that they can directly or indirectly transmit information from one to the other are considered to be "in electronic communication" with one another.
  • a computer network containing multiple computers may have a central computer (“central node") that processes information to one or more sub- computers that carry out specific tasks (“sub-nodes”).
  • Some networks comprises computers that are in "different geographic locations" from one another, meaning that the computers are located in different physical locations (i.e., aren't physically the same computer, e.g., are located in different countries, states, cities, rooms, etc.).
  • detection assay component refers to a component of a system capable of performing a detection assay.
  • Detection assay components include, but are not limited to, hybridization probes, buffers, and the like.
  • a detection assay configured for target detection refers to a collection of assay components that are capable of producing a detectable signal when carried out using the target nucleic acid.
  • a detection assay that has empirically been demonstrated to detect a particular single nucleotide polymo ⁇ hism is considered a detection assay configured for target detection.
  • unique detection assay refers to a detection assay that has a different collection of detection assay components in relation to other detection assays located on the same detection panel.
  • a unique assay doesn't necessarily detect a different target (e.g. SNP) than other assays on the same detection panel, but it does have a least one difference in the collection of components used to detect a given target (e.g. a unique detection assay may employ a probe sequences that is shorter or longer in length than other assays on the same detection panel).
  • the term “candidate” refers to an assay or analyte, e.g., a nucleic acid, suspected of having a particular feature or property.
  • a “candidate sequence” refers to a nucleic acid suspected of comprising a particular sequence
  • a “candidate oligonucleotide” refers to an oligonucleotide suspected of having a property such as comprising a particular sequence, or having the capability to hybridize to a target nucleic acid or to perform in a detection assay.
  • a “candidate detection assay” refers to a detection assay that is suspected of being a valid detection assay.
  • the term “detection panel” refers to a substrate or device containing at least two unique candidate detection assays configured for target detection.
  • valid detection assay refers to a detection assay that has been shown to accurately predict an association between the detection of a target and a phenotype (e.g. medical condition).
  • valid detection assays include, but are not limited to, detection assays that, when a target is detected, accurately predict the phenotype medical 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99,9% of the time.
  • Other examples of valid detection assays include, but are not limited to, detection assays that quality as and/or are marketed as Analyte-Specific Reagents (i.e. as defined by FDA regulations) or In- Vitro Diagnostics (i.e. approved by the FDA) .
  • kits refers to any delivery system for delivering materials.
  • delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another.
  • reaction reagents e.g., oligonucleotides, enzymes, etc. in the appropriate containers
  • supporting materials e.g., buffers, written instructions for performing the assay etc.
  • kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials.
  • fragment kit refers to a delivery systems comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately.
  • a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides.
  • fragment kit is intended to encompass kits containing Analyte specific reagents (ASR's) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.”
  • a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components).
  • kit includes both fragmented and combined kits.
  • the term "information” refers to any collection of facts or data. In reference to information stored or processed using a computer system(s), including but not limited to internets, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.).
  • information related to a subject refers to facts or data pertaining to a subject (e.g., a human, plant, or animal).
  • genomic information refers to information pertaining to a genome including, but not limited to, nucleic acid sequences, genes, allele frequencies, RNA expression levels, protein expression, phenotypes conelating to genotypes, etc.
  • Allele frequency information refers to facts or data pertaining allele frequencies, including, but not limited to, allele identities, statistical cone ⁇ ations between the presence of an allele and a characteristic of a subject (e.g., a human subject), the presence or absence of an allele in a individual or population, the percentage likelihood of an allele being present in an individual having one or more particular characteristics, etc.
  • assay validation information refers to genomic information and/or allele frequency information resulting from processing of test result data (e.g. processing with the aid of a computer). Assay validation information may be used, for example, to identify a particular candidate detection assay as a valid detection assay.
  • Coupled refers to attachments between objects that do not, by themselves, provide a pressure-tight seal.
  • two metal plates that are attached by screws or pins may comprise a coupled attachment. While the two plates are attached, the seam between them does not form a pressure-tight seal (i.e., gas and/or liquid can escape through the seam).
  • synthesis and purge component refers to a component of a synthesizer containing a cartridge for holding one or more synthesis columns attached to or connected to a drain plate for allowing waste or wash material from the synthesis columns to be directed to a waste disposal system.
  • cartridge refers to a device for holding one or more synthesis columns.
  • cartridges can contain a plurality of openings (e.g., receiving holes) into which synthesis columns may be placed.
  • Rotary cartridges refer to cartridges that, in operation, can rotate with respect to an axis, such that a synthesis column is moved from one location in a plane (a reagent dispensing location) to another location in the plane (a non-reagent dispensing location) following rotation of the cartridge.
  • nucleic acid synthesis column refers to a container or chamber in which nucleic acid synthesis reactions are carried out.
  • synthesis columns include plastic cylindrical columns and pipette tip formats, containing openings at the top and bottom ends.
  • the containers may contain or provide one or more matrices, solid supports, and/or synthesis reagents necessary to carry out chemical synthesis of nucleic acids.
  • synthesis columns contain a solid support matrix on which a growing nucleic acid molecule may be synthesized.
  • Nucleic acid synthesis columns may be provided individually; alternatively, several synthesis columns may be provided together as a unit, e.g., in a strip or anay, or as device such as a plate having a plurality of suitable chambers.
  • Columns may be constructed of any material or combination of materials that do not adversely affect (e.g., chemically) the synthesis reaction or the use of the synthesized product.
  • columns or chambers may comprise polymers such as polypropylene, fiuoropolymers such as TEFLON, metals and other materials that are substantially inert to synthesis reaction conditions, such as stainless steel, gold, silicon and glass.
  • chambers comprise a coating of such a suitable material over a structure comprising a different material.
  • seal refers to any means for preventing the flow of gas or liquid through an opening.
  • a seal may be formed between two contacted materials using grease, o-rings, gaskets, and the like.
  • one or both of the contacted materials comprises an integral seal, such as, e.g., a ridge, a lip or another feature configured to provide a seal between said contacted materials.
  • An "airtight seal” or “pressure tight seal” is a seal that prevents detectable amounts of air from passing through an opening.
  • a “substantially airtight” seal is a seal that prevents all but negligible amounts of air from passing through an opening.
  • Negligible amounts of air are amounts that are tolerated by the particular system, such that desired system function is not compromised.
  • a seal in a nucleic acid synthesizer is considered substantially airtight if it prevents gas leaks in a reaction chamber, such that the gas pressure in the reaction chamber is sufficient to purge liquid in synthesis columns contained in the reaction chamber following a synthesis reaction. If gas pressure is depleted by a leak such that synthesis columns are not purged (e.g., resulting in overflow during subsequent synthesis rounds), then the seal is not a substantially airtight seal.
  • a substantially airtight seal can be detected empirically by carrying out synthesis and checking for failures (e.g., column overflows) during one or a series of reactions.
  • sealed contact point refers to sealed seams between two or more objects. Seals on sealed contact points can be of any type that prevent the flow of gas or liquid through an opening. For example, seals can sit on the surface of a seam (e.g., a face seal) or can be placed within a seam, such that a circumferential contact is created within the seam.
  • alignment detector refers to any means for detecting the position of an object with respect to another object or with respect to the detector.
  • alignment detectors may detect the alignment of a dispensing end of a dispensing device (e.g., a reagent tube, a waste tube, etc.) to a receiving device (e.g., a synthesis column, a waste valve, etc.).
  • Alignment detectors may also detect the tilt angle of an object (e.g., the angle of a plane of an object with respect to a reference plane). For example, the tilt angle of a plate mounted on a shaft may be detected to ensure a proper pe ⁇ endicular relationship between the plate and the shaft.
  • Alignment detectors include, but are not limited to, motion sensors, infra-red or LED- based detectors, and the like.
  • Alignment markers refers to reference points on an object that allow the object to be aligned to one or more other objects.
  • Alignment markers include pictorial markings (e.g., anows, dots, etc.) and reflective markings, as well as pins, raised surfaces, holes, magnets, and the like.
  • motor connector refers to any type of connection between a motor and another object.
  • a motor designed to rotate another object may be connected to the object through a metal shaft, such that the rotation of the shaft, rotates the object.
  • the metal shaft would be considered a motor connector.
  • packing material refers to material placed in a passageway (e.g., a synthesis column) in a manner such that it provides resistance against a pressure differential between the two ends of the passageway (i.e. hinders the discharge of the pressure differential).
  • Packing material may comprise a single material or multiple materials.
  • packing material comprising a nucleic acid synthesis matrix (e.g., a solid support for nucleic acid synthesis such as controlled pore glass, polystyrene, etc.) and/or one or more frits are used in synthesis columns to maintain a pressure differential between the two ends of the synthesis column.
  • Packing material may be distributed into the reaction chambers in a variety of forms.
  • synthesis support matrix may be provided as a granular powder.
  • support matrix may be provided in a "pill" form, wherein an appropriate amount of a support material is held together with a binder to form a pill, and wherein one or more pills are provided to a reaction chamber, as appropriate for the scale of the intended reaction, and further wherein the binder is removed or inactivated (e.g., during a wash step) to allow the powdered matrix to function in the same manner as an unbound powder.
  • pill embodiment provides the advantages of facilitating the process of pre-measuring synthesis support materials, allowing easy storage of support matrices in a pre-measured form, and simplifying provision of measured amounts of synthesis support matrix to a reaction chamber.
  • the term "idle,” in reference to a synthesis column, refers to columns that do not take part in a particular synthesis reaction step of a nucleic acid synthesizer.
  • Idle synthesis columns include, but are not limited to, columns in which no synthesis occurs at all, as well as columns in which synthesis has been completed (e.g., for short oligonucleotide) while other columns are actively undergoing additional synthesis steps (e.g., for longer oligonucleotides).
  • active in reference to a synthesis column, refers to columns that take part (or are taking part) in a particular synthesis reaction step of a nucleic acid synthesizer. Active synthesis columns include, but are not limited to, columns in which liquid reagents are being dispensed into, or columns that contain liquid reagents (e.g. waiting to be purged), or columns that are in the process of being purged.
  • an O-ring refers to a component having a circular or oval opening to accommodate and provide a seal around another component liaving a circular or oval external cross-section.
  • An O-ring will generally be composed of material suitable for providing a seal, e.g., a resilient air-or moisture-proof material.
  • an O-ring may be a circular opening in a larger gasket.
  • a single gasket may contain multiple openings and thus provide multiple O-rings.
  • an O-ring may be ring-shaped, i.e., it may have circular interior and exterior surfaces that are essentially concentric.
  • the term "viewing window” refers to any fransparent component configured to allow visual inspection of an item or material through the window.
  • An enclosure may include a transparent portion that provides a viewing window for item within the disclosure.
  • an enclosure may be made entirely of a transparent material. In such embodiments, the entire enclosure can be considered a viewing window.
  • a "viewing window” in an enclosure that is “configured to allow visual inspection” of items in the enclosure “without opening the enclosure” refers to a viewing window in an enclosure of sufficient size, location, and fransparency to allow the item to be viewed, unhindered, by the human eye.
  • the window is configured to allow viewing of the reagents bottles by the human eye to determine if the bottles or full or empty.
  • a window that does not provide adequate visual inspection of each of the reagent bottles is not configured to allow visual inspection of reagents in the enclosure without opening the enclosure.
  • the term "enclosure” refers to a container that separates materials contained in the enclosure from the ambient environment (e.g., as in a sealed system).
  • an enclosure may be used with a reagent station to contain reagents within an interior chamber of the enclosure, and therefore separate the reagents from the ambient environment.
  • the enclosure provides an airtight or substantially airtight seal between the interior and exterior of the enclosure.
  • the enclosure may contain one or more valves (e.g., ventilation ports), doors, or other means for allowing gasses or other materials (e.g., reagent bottles) to enter or leave the interior environment of the enclosure.
  • reaction enclosure refers to an enclosure that separates the reaction columns or other reaction vessels (e.g., microplates) from the ambient environment.
  • a chamber bowl 18 closed with a top cover 30 and sealed with a chamber seal 31 is one exemplary embodiment of a reaction enclosure.
  • a reaction enclosure is a synthesis case, e.g., as provided with a POLYPLEX synthesizer (GeneMachines, San Carlos, CA) and with the synthesizers described in WO 00/56445.
  • reaction enclosures can be sealed during at least one step of operation (e.g., during active synthesis) and can be opened for at least one step of operation (e.g., for inserting or removing reaction vessels).
  • the term "top enclosure” refers to an enclosure that forms a primarily enclosed space over the top cover.
  • the top enclosure has four sides (e.g., four top enclosure sides, e.g., 98) and a top panel (e.g., 97) that form a primarily enclosed space (e.g.
  • the primarily enclosed space (e.g., 104) is open to the ambient environment through a ventilation slot (e.g., 100) in the top cover or the top enclosure.
  • the top panel e.g., 99
  • an outer window e.g., 101
  • the combination of a “top enclosure” and “top cover” is refened to collectively as the “lid enclosure”.
  • the "lid enclosure” e.g., 102
  • the top cover e.g., 30
  • the top panel serving as the surface opposite the top cover
  • the four side walls being the top enclosure sides (e.g., 98).
  • the lid enclosure is hinged so that is may be moved upward and downward.
  • primarily enclosed space refers to a space having reduced contact with the ambient environment. A primarily enclosed space need not be sealed.
  • a primarily enclosed space 104 of a lid enclosure of the present invention has contact with the ambient environment through a ventilation slot (e.g., 100).
  • a primarily enclosed space 104 of a synthesizer base 2 has contact with the ambient environment through a ventilation slot (e.g., 100)
  • the term "ventilated workspace” refers to a work area that is open to the ambient environment but that is maintained under negative air pressure such that air flows into the ventilated workspace, thereby reducing or preventing the flow of fumes and emissions from the ventilated workspace into the ambient environment.
  • a ventilated workspace is a fume hood (e.g. a chemical fume hood).
  • the ventilated workspace that is part of an apparatus (e.g., a nucleic acid synthesizer), such that the negative air pressure is maintained over a reaction chamber to draw air away from the reaction chamber so as to prevent the air from entering the ambient environment.
  • synthesis refers to the assembly of polymers from smaller units, such as monomers.
  • fluid connection refers to a continuous fluid path between components.
  • reaction support refers to a structure supporting, comprising, or containing one or more reaction chambers.
  • the term "rare mutation” refers to a mutation that is present in 20% or less (preferably 10% or less, more preferably 5% or less, and more preferably 1% or less) of a population of nucleic acid molecules in a sample (i.e., wherein the remaining 80% or more of the nucleic acid molecules have a wild type sequence or a different mutation in the conesponding region of the nucleic acid molecules).
  • the term "distinct" in reference to signals refers to signals that can be differentiated one from another, e.g., by spectral properties such as fluorescence emission wavelength, color, absorbance, mass, size, fluorescence polarization properties, charge, etc., or by capability of interaction with another moiety, such as with a chemical reagent, an enzyme, an antibody, etc.
  • the present invention relates to detection assay development, production, usage and optimization.
  • the present invention provides systems and methods for acquiring and analyzing biological information.
  • the present invention also provides detection assay production with improved oligonucleotide synthesis and processing systems.
  • the present invention further provides systems that integrate biological information collection with detection assay production that allow for rapid development of commercial products, such as analyte specific reagents (ASRs) and in vitro diagnostics (IVDs).
  • ASRs analyte specific reagents
  • IVTDs in vitro diagnostics
  • the present invention provides systems and methods for the use of genetic information in the generation of assays for detecting the genetic identity of samples, the production of assays, the use of assays for gathering genetic information of individuals and populations, and the storage, analysis, and use of the obtained information, including the use of information in selecting detection assays for research use, use in panels, use as ASRs, and use in clinical diagnostics (e.g., in vitro diagnostics).
  • the present invention provides systems and methods for analyzing available sequence information (e.g., publicly available sequence information and information obtained by the methods described herein) in the selection of informative DNA and RNA target sequences for detections and analysis of individuals and populations.
  • available sequence information e.g., publicly available sequence information and information obtained by the methods described herein
  • the present invention also provides systems and methods for the design and production of detection assays directed to such target sequences.
  • the present invention further provides systems and methods for the collection, storage and analysis of data derived from detection assays.
  • the present invention provides integrated systems and methods that exploit the synergies of the above systems and methods to provide comprehensive solutions, allowing for large scale and informative analysis of sequences for identifying genotype/phenotype conelations, measuring differences in gene expression, identifying allele frequencies in populations, and typing individuals and populations for important (e.g., medically relevant) sequences.
  • the present invention applies data obtained from detection assays to improve the selection of target sequences, design of improved assays, and selection of assays that are suitable for use on multi-analyte panels, as ASRs, and for clinical diagnostics.
  • the present invention provides detection assay development, production and optimization (See, section A below). For example, orders are received from customer (e.g. a target sequence is entered via a web interface), and the orders are processed (See, section A.I., "Target Sequence Selection), and Detection Assays are Designed (See Section A.III, below).
  • the designed assays are produced (or filled from inventory) in a production facility (See, section III below).
  • the assays that are produced are stored in inventory or shipped to customers.
  • each of these components are operably linked to a central data management system (e.g. running ente ⁇ rise software such as Oracle), such that data and status of orders is communicated throughout the system (See, Section A.1V., below).
  • a central data management system e.g. running ente ⁇ rise software such as Oracle
  • Detection assays are shipped to customers who use the detection assay and generate data.
  • the data generated by the use of these detection assays is gathered, analyzed, and stored (See, section AN, below). This information may then be integrated with the order, design, production and storage components mentioned above (See, ANI. below).
  • data is continuously generated that allows, for example, an association between detection assays or targets with particular medical conditions to be established. Gathering, analyzing, and producing detection assays while generating association data allows the clinical detection assays (e.g., ASRs and In vitro Diagnostics) to be developed and validated (See, Section, B below) through a funneling process that allows a business to focus on particularly useful assays.
  • ASRs e.g., ASRs and In vitro Diagnostics
  • Assays may be inco ⁇ orated in panels or databases in order to be distributed to research facilities (e.g. ASR certified), hospitals, doctors, and other customers (See, Section, C below).
  • Integrating the production systems, databases, and managements systems of the present invention allows efficient production of particular assays, as well as rapid identification of ASRs, and in vitro diagnostics. Furthermore, integration of these systems allows for accurate business pricing of various assays (See, section C, below), allowing, for example, differential pricing of ASRs and In Vitro Diagnostics.
  • the discussion provides a description of certain prefened illustrative embodiments of the present invention and is not intended to limit the scope of the present invention.
  • the discussion focuses on the application of the present invention to the detection of DNA targets, but it should be understood that the methods and systems are intended for use in the development of tools for the analysis of any nucleic acid analyte, e.g., DNA or RNA.
  • the discussion often focuses on the characterization of SNPs using INVADER assay technology. It should be understood that the methods and systems of the present invention are intended for use in detecting other biologically relevant factors using a wide variety of detection assay technologies.
  • the present invention provides systems and methods for developing detection assays for research and clinical use.
  • detection assay development, production, and optimization is illustrated below for hybridization-bases assays.
  • One skilled in the art will appreciate the general applicability of various aspects of this description to other types of detection assays.
  • the discussion of detection assay development, production, and optimization is provided in the following sections: I) Target Sequence Selection; II) Detection Assay Design; III) Detection Assay Production; IV) Data Management Systems; V) Detection Assay Use and Data Generation and Collection; and VI) Integrated Information, Design, and Production (Optimization). It will be appreciated that every step may not be required for each detection assay. For example, where a valid target sequence and assay design are already known, production and testing may be started directly.
  • the steps may be used for original assay development and/or may be used to re-evaluate a pre-existing detection assay, whether is be for a research or a clinical detection assay.
  • Examples of process configurations for integrating the steps are provided in Figures 1, 58, 61, and 62.
  • direct clients or distributors go through an order entry process (described in detail below).
  • Detections assays conesponding to particular oligonucleotides, primers, panels, polymo ⁇ hisms e.g., SNPs
  • assay design software e.g.,
  • INVADERCREATOR software If a request conesponds to a previously validated or ordered sequences, software locates the product and proceeds with the order accordingly. Designed detection assays are then sent to a production facility for production and validation (described in detail below). Data generated by the process or from use of the detection assays and collected and stored in databases (described in detail below).
  • the ability to detect the presence or absence of specific target sequences in a sample underlies much of the fields of molecular diagnostics and molecular medicine. For example, tremendous effort has been expended in the development of detection assays for nucleic acid sequence mutations that conelate to phenotypes of interest (e.g., inherited diseases). During the development of the present invention, it was found that the design of a detection assay based on a published target sequence was often not sufficient to produce viable assays. In some circumstances assays will not work at all. In others, they may work for particular individuals or populations, but fail with other individuals or populations. The present invention provides systems and methods for selecting appropriate target sequences that can be successfully targeted by detection assays.
  • oligonucleotide is designed to hybridize to a portion of the target sequence; the presence of the hybrid, or the cleavage, elongation, ligation, disassociation, or other alterations of the oligonucleotide are detected as a means for characterizing the presence or absence of the sequence of interest (e.g., a SNP). Because there is sequence heterogeneity in the population, an oligonucleotide designed to hybridize to a target sequence of one individual may not hybridize to the conesponding sequence from another individual.
  • a first individual may have a gene sequence containing a SNP that is to be detected.
  • a second individual may have the SNP, but also may have additional sequence differences in the vicinity of the SNP that prevent the hybridization of an oligonucleotide that was designed based on the sequence of the first individual.
  • target sequence information obtained from a public source may contain enors (e.g., may provide the wrong sequence) or may comprise incomplete, but essential, information.
  • a given target sequence may be found in multiple locations in the genome — the intended region that the assay is designed to detect, and unintended regions that would result in false positive or otherwise misleading assay results.
  • the systems and methods of the present invention provide an analysis of candidate target sequences to determine if they are suitable for use in detection assays.
  • the systems and methods of the present invention also select appropriate sequences that are likely to function in the intended detection assay.
  • This aspect of the present invention is refened to herein as "in silico analysis," as computer analysis is conducted to analyze candidate target sequences against sequence and sequence-related information databases. In silico analysis may be performed prior to, or in conjunction with other processes of the present invention (e.g., detection assay design and production, selection of materials for panels, ASRs, and clinical tests, etc.).
  • In silico analysis methods of the present invention include one or more of the following sequence analysis and processing steps: input of a candidate sequence; editing of the candidate sequence, where necessary; screening of the candidate sequence for repeat sequences; screening of the candidate sequence for research artifact sequences; identification of the candidate sequence in a sequence database; conformation of the candidate sequence in a second (or additional) sequence database; information gathering using one or more sequence information databases; problem reporting; and/or transmission of an approved target sequence for production (e. g. , automated production) .
  • Sequences may be input for in silico analysis from any number of sources.
  • sequence information is entered into a computer.
  • the computer need not be the same computer system that carries out in silico analysis.
  • candidate target sequences may be entered into a computer linked to a communication network (e.g., a local area network, Internet or Intranet).
  • a communication network e.g., a local area network, Internet or Intranet.
  • users anywhere in the world with access to a communication network may enter candidate sequences at their own locale.
  • a user interface is provided to the user over a communication network (e.g., a World Wide Web-based user interface), containing entry fields for the information required by the in silico analysis (e.g., the sequence of the candidate target sequence).
  • the user interface can ensure that the user inputs the requisite amount of information in the conect format.
  • the user interface requires that the sequence information for a target sequence be of a minimum length (e.g., 20 or more, 50 or more, 100 or more nucleotides) and be in a single format (e.g., FASTA).
  • the information can be input in any format and the systems and methods of the present invention edit or alter the input information into a suitable form for in silico analysis.
  • the systems and methods of the present invention search public databases for the short sequence, and if a unique sequence is identified, convert the short sequence into a suitably long sequence by adding nucleotides on one or both of the ends of the input target sequence.
  • sequence information is entered in an undesirable format or contains extraneous, non-sequence characters, the sequence can be modified to a standard format (e.g., FASTA) prior to further in silico analysis.
  • the user interface may also collect information about the user, including, but not limited to, the name and address of the user.
  • target sequence entries are associated with a user identification code.
  • sequences are input directly from assay design software (e.g., the INVADERCREATOR software described below).
  • each sequence is given an ID number.
  • the ID number is linked to the target sequence being analyzed to avoid duplicate analyses. For example, if the in silico analysis determines that a target sequence conesponding to the input sequence has already been analyzed, the user is informed and given the option of by-passing in silico analysis and simply receiving previously obtained results.
  • the customer order component also includes one or more screens or web pages that include detection assay instrumentation data.
  • Detection assay instrumentation data includes data describing various systems and devices, including but not limited to liquid handlers, workstations, and other automation options shown in, for example, Table 2, which are used to facilitate use of the detection assays created using the methods and systems described herein.
  • a customer selects a particular type of panel format, e.g. 96 well, 385 well or 1536 well and assay configuration, he is automatically linked or presented with data of appropriate conesponding devices that are used to read the panel format which are offered for sale to the customer.
  • the system stores information about the type of instrumentation the customer already has in house or has previously purchased, and automatically determines and suggests the type of panel format for detection assays that the customer should buy on the customer order component, e.g. 96 well, 384 well or 1536 well.
  • the customer is also provided with instrumentation pricing data, instrument specification data, delivery data, shipping data, for various combinations of instrumentation that would suit the customer's needs.
  • the customer order entry component can then feed data on the customer's instrumentation order (or in-house instrumentation where the customer makes a selection from an instrumentation menu presented on the web site) to the detection assay production component (including resident hardware and software components thereof) so that projections can be made as to the number and type of various detection assay starting materials that need to be purchased or stocked based upon the customers selection of instrumentation and projected usage of disposable detection assays, e.g. reagents, glass slides, plastic arrays, etc.
  • a single customer's (or a plurality of networked customers') instrumentation has a communication link to the customer order component or the detection assay production facility for exchanging data therebetween.
  • detection assay usage data is transfened from the customer's instrumentation to the detection assay production facility (or other components of the system) to help schedule and produce detection assays and order reagents and components therefore, or prompt the customer via e-mail that his stock of detection assays is nearing a predetermined number and that the customer needs to re-order detection assays.
  • the customer's instrumentation automatically sends order data to the customer order component or other component of the system automatically ordering additional detection assays for one or more customers.
  • these systems are linked to a pricing component, wherein repeat customers may receive beneficial pricing for re-orders or upon reaching a total threshold volume of orders over time.
  • an ordering and information system of the present invention is connected to a public network to allow any user access to the information.
  • private electronic communication networks are provided. For example, where a customer or user is a repeat customer (e.g., a distributor or large diagnostic laboratory), the full-time dedicated private connection may be provided between a computer system of the customer and a computer system of the systems of the present invention. The system may be arranged to minimize human interaction.
  • inventory control software is used to monitor the number and type of detection assays in possession of the customer.
  • a query is sent at defined intervals to determine if the customer has the appropriate number and type of detection assay, and if shortages are detected, instructions are sent to design, produce, and/or deliver additional assays to the customer.
  • the system also monitors inventory levels of the seller and in prefened embodiments, is integrated with production systems to manage production capacity and timing.
  • a user-friendly interface is provided to facilitate selection and ordering of detection assays. Because of the hundreds of thousands of detection assays available and or polymo ⁇ hisms that the user may wish to intenogate, the user-friendly interface allows navigation through the complex set of options.
  • the first layer provides a display of all of the chromosomes of an organism. The user selects the chromosome or chromosomes of interest. Selection of the chromosome provides a more detailed map of the chromosome, indicating banding regions on the chromosome. Selection of the desired band leads to a map showing gene locations.
  • One or more additional layers of detail provide base positions of polymo ⁇ hisms, gene names, genome database identification tags, annotations, regions of the chromosome with pre-existing developed detection assays that are available for purchase, regions where no pre-existing developed assays exist but that are available for design and production, etc.
  • Selecting a region, polymo ⁇ hism, or detection assay takes the user to an ordering interface, where information is collected to initiate detection assay design and/or ordering.
  • a search engine is provided, where a gene name, sequence range, polymo ⁇ hism or other query is entered to more immediately direct the user to the appropriate layer of information.
  • a user may select a PCR (or other amplification technology) or non-PCR option, depending if they want to employ amplification along with their detection assay.
  • the PCR primer section may be employed to design such assays, taking into consideration the target and the detection assay selected by the user (see below).
  • the ordering, design, and production systems are integrated with a finance system, where the pricing of the detection assay is determined by one or more factors: whether or not design is required, cost of goods based on the components in the detection assay, special discounts for certain customers, discounts for bulk orders, discounts for re-orders, price increases where the product is covered by intellectual property or contractual payment obligations to third parties, and price selection based on usage.
  • pricing is increased.
  • the pricing increase for clinical products occurs automatically.
  • the systems of the present invention are linked to FDA, public publication, or other databases to determine if a product has been certified for clinical diagnostic or ASR use.
  • the system and method of the present invention includes an organism-specific web order entry component.
  • the organism-specific web order entry component comprises one or more screens and/or linked web pages that are interactively directed to present for sale one or more detection assays for a specific organism(s).
  • a web page or combination of web pages provides displays of the chromosomes, genes, and/or detection assays for various transgenic plants, wild type plants, wild type animals, transgenic animals, and/or genetically altered or naturally occurring microorganisms, e.g. bacteria, viruses, etc.
  • one or more screens of different linked web pages permit a user to drill down into a specific genus, species and/or sub-species of an organism and/or chromosomes (or sub-parts thereof), and display the various detection assays created for the organism and/or detection assays that have been created that may be used across various organisms.
  • the detection assays are optionally linked to specific genes or portions of chromosomes of a single organism or of multiple related or unrelated organisms.
  • databases and software for in silico analysis utilizes one or more sequence and information databases (e.g., public or private sequence databases) and software applications for processing sequence and database information (See, e.g. Figure 3),
  • databases and software for in silico analysis are housed in a single location on one or more computers. Housing the databases and processing software locally provides increased and consistent speed and access to information.
  • one or more databases and software components located on external computers are accessed over a communication network (e.g., accessed over the World Wide Web).
  • databases that are maintained locally are updated regularly
  • a new version is downloaded to local servers.
  • databases are surveyed periodically to determine if a new version is available and, if so, one is downloaded.
  • more than one copy of each database is available locally.
  • downloaded data is parsed to extract the data, and the parsed data is configured to automatically populate the fields of one or more receiving databases (e.g., an association database, a SNP database).
  • Perl scripts are used to sort data, e.g., line-by-line, and to create new text files (e.g., having data tagged according to the receiving field in the receiving database) for importation into the fields of a receiving database.
  • the database analysis system comprises one or more central nodes
  • the central node controls the flow of information between sub-nodes, sending search requests to the sub-nodes and receiving search results from the sub- nodes. For example, in some embodiments, the central node directs data (e.g., candidate target sequence) to a sub node for a database search, receives the results, and directs the information to another sub-node for additional database searching. In some prefened embodiments, the central node directs information to multiple sub nodes simultaneously (e.g., for multiple concunent database searches).
  • data e.g., candidate target sequence
  • the central node directs information to multiple sub nodes simultaneously (e.g., for multiple concunent database searches).
  • individual databases are split among multiple (e.g., two) sub-nodes.
  • databases are housed on a single node.
  • databases are present in multiple copies on multiple sub- nodes.
  • the central node monitors database load and status on each sub-node and directs searches to the node with the greatest available capacity.
  • the central node further directs resource management software. For example, individual nodes are sent test sequences on a regular basis to ensure that they are receiving information and processing information on a desired time scale.
  • the central node directs information to a secondary sub node containing a copy of the database.
  • sub-nodes conduct self- monitoring routines and send status reports back to the central node, For example, in some embodiments, if a search on a sub-node fails or times out, the sub-node reports this information back to the central node so that appropriate action can be taken (e.g., send the search to another node and/or flag a particular sub-node for intervention).
  • the central node maintains a queue of jobs submitted to each sub-node and warns human supervisors if a job fails to be completed.
  • the central node comprises one or more workstations.
  • the sub nodes comprise two or more workstations.
  • the sub nodes comprise 5 or more workstations.
  • the sub nodes comprise 10 or more workstations.
  • the present invention is not limited to a particular model or type of workstation. One skilled in the art understands that a variety of new processors of increasing speeds are regularly introduced into the market and that any suitable work station may be substituted for those described herein.
  • in silico analysis of a candidate target sequence is completed in less than 10 seconds. In some prefened embodiments, in silico analysis of a candidate target sequence is completed in less than 2 seconds. In still more prefened embodiments, in silico analysis is completed in less than one second. In some embodiments, more than one (e.g., at least 5, preferably at least 20, and even more preferably, at least 100) sequences are analyzed simultaneously using the in silico analysis system of the present invention.
  • the first step of in silico analysis of candidate target sequences is prescreening the candidate target sequences to maximize sequence database search efficiency.
  • candidate target sequences are searched for repeat sequences.
  • "Repeat sequences" refers to sequences that are known to repeat multiple times in a sample (e.g., in an organism's genome). Many genomes contain large regions of repeated sequences. The presence of repeated sequences in detection assay hybridization oligonucleotides can cause the oligonucleotide to hybridize to sequences other than, and/or in addition to, the intended target. Additionally, because repeat sequences are found in multiple copies in the genome, databases searches may operate very slowly or may not proceed.
  • RepeatMasker is a perl script used in conjunction with REPBASE, which is a database of known Human repeats and is used to screen for repeat sequences.
  • REPBASE is a database of known Human repeats and is used to screen for repeat sequences.
  • Repeat Masker screens DNA sequences for interspersed repeats and low complexity DNA sequences. Sequence information in FASTA format is input through a web-browser interface or by uploading a file. Multiple sequences may be input at once or may be contained within a file. There is no limit to the' length of the query sequence or size of the batch file. Sequence comparisons in RepeatMasker are performed by the program Cross-match, an implementation of the Smith- Waterman-Gotoh algorithm developed by Phil Green. In some embodiments, RepeatMasker is run using MaskerAid (Bioinformatics
  • MaskerAid allows the faster WU-BLAST search engine to substitute transparently for CrossMatch, yielding speed improvement while effectively maintaining sensitivity.
  • MaskerAid is fundamentally a software "wrapper" around WU-BLAST that makes it appear and function very much like CrossMatch.
  • the output of the program is an annotation of the repeats that are present in the sequence of interest as well as a modified version of the sequence in which all the annotated repeats have been masked.
  • the program returns three or four output files for each query.
  • One contains the submitted sequence(s) in which all recognized interspersed or simple repeats have been masked. In the masked areas, each base is replaced with an N, so that the returned sequence is of the same length as the original.
  • a table annotating the masked sequences as well as a table summarizing the repeat content of the query sequence is returned.
  • a file with alignments of the query with the matching repeats is returned as well.
  • Regions of low complexity like simple tandem repeats, polypurine and AT-rich regions can lead to spurious matches in database searches.
  • they are masked along with the interspersed repeats.
  • Do not mask simple only interspersed repeats are masked. This may, for example, be prefened in some embodiments where the masked sequence will be analyzed by a gene prediction program.
  • Only mask simple one can mask only the low complexity regions (e.g., in some embodiments in which it is desirable to quickly locate polymo ⁇ hic simple repeats in a sequence).
  • the repeat sequences are replaced by Xs instead of Ns. This allows one to distinguish the masked areas from possibly existing ambiguous sequences or other stretches of Ns in the original sequence.
  • the use of X, N, or both may be desired for compatibility with database search engines used in the subsequent steps of the in silico analysis. .
  • only the masked candidate target sequence is used in further in silico analysis.
  • both the masked and unmasked sequences are used in subsequent searches.
  • a majority or the entirety of the candidate target sequence may be masked by RepeatMasker. When this occurs, in some embodiments, a warning is sent to the user indicating that a potentially undesirable amount of the target sequence comprises repeat sequence.
  • the user is then give the option of selecting a different target sequence or proceeding with the original sequence (or electing both options).
  • a decision to proceed with the sequence is selected, an unmasked version of the sequence is processed through the remaining in silico analysis steps. Where there is a portion of the original candidate target sequence that is not masked, both unmasked and masked sequences may be processed through the remaining in silico analysis steps.
  • in silico analysis is discontinued and the candidate target sequence is sent to production (Section III, below).
  • an analysis is performed to determine if the candidate target sequence contains undesired artifact sequences.
  • a number of sequences deposited in public databases contain vector sequence or other sequence artifacts as a result of molecular biology handling during their initial isolation and characterization. These artifact sequences often represent synthetic sequences not conesponding to a genome sequence, or inappropriately conesponding to a genome sequence other than the intended target. Where candidate target sequences are selected that contain artifact sequences, they are more likely to fail in detection assays and are more likely to result in undesirably long search times during the remaining in silico analysis steps. For example, rather than representing a sequence that appears once in a human genome, artifact sequence may conespond to thousands of deposited database sequence that each mistakenly contain a common vector sequence.
  • the present invention employs VecScreen (available at the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health public web site).
  • VecScreen provides a system for identifying segments of a nucleic acid sequence that may be of vector origin.
  • VecScreen searches a query for segments that match any sequence in a specialized non-redundant vector database (UniVec).
  • the search uses a BLAST search routine with parameters preset for optimal detection of vector contamination. Those segments of the query that match vector sequences are categorized according to the strength of the match, and their locations are displayed.
  • the sequence of any vector contamination should theoretically be identical to the known sequence of the vector. In practice, occasional differences are expected to arise from sequencing enors, and less frequently, from engineered variants or spontaneous mutations.
  • the search parameters used for VecScreen are chosen to find sequence segments that are identical to known vector sequences or which deviate only slightly from the known sequence. Vector containing sequences identified are then masked.
  • the Repeat Masker and VecScreen screening are combined into a single search.
  • the candidate target sequence is first screened by VecScreen, with the results then passed through Repeat Masker. Once the screening is complete, masked sequences and/or unmasked sequences are ready for database searching as described below.
  • database searches are performed on the candidate target sequences.
  • Databases searches are used, among other pu ⁇ oses, to corrfirm that 1) the candidate target sequence is a sequence conesponding to a known sequence, 2) the candidate target sequence conesponds to a unique sequence in the sample to be tested, and 3) the candidate target sequence conesponds to a reliable (e.g., confirmed) sequence.
  • the database searches are also used to gather information (allele frequencies, disease associations, variants, location in a genome, associated patents and patent applications, etc.) about the candidate target sequence.
  • the output information from the database searches is stored in a file associated with the candidate target sequence. In further embodiments, the output information is displayed to the user.
  • the present invention is not limited to the databases disclosed herein. Any database that provides relevant information may find use in the searches of the present invention.
  • searches are performed consecutively. In other embodiments, searches are performed concunently. In prefened embodiments, some searches are performed consecutively and others are performed concunently.
  • searches are performed using BLAST (Basic Local Alignment Search Tool) search mode using FASTA formatted sequences.
  • results from database searches are output as text files. Results are then converted to a format that is suitable for import into an Oracle database.
  • the Bio Java Project is used to convert text output into an XML-like stream that is then inco ⁇ orated into an Oracle database.
  • rat, mouse or any other organism sequence databases Other databases that are searched or used in or with various components of the invention include rat, mouse or any other organism sequence databases. It is also appreciated that the present invention can cross reference detection assays across different species of organisms. By way of example, if a customer designates a human detection assay on a customer order entry screen, the software or routines of the invention may automatically present and offer for sale on the customer's computer screen the same or similar detection assay for rats, mice or any other organism.
  • candidate target sequences are first used to search several databases which catalog SNPs.
  • the targeted databases include NCBFs dbSNP, the UK's HGBASE SNP database, the SNP Consortium database, and the Japanese Millenium Project's SNP database.
  • the dbSNP database serves as a cenfral repository for both single base nucleotide substitutions and short deletion and insertion polymo ⁇ hisms, and includes all the SNPs identified in the SNP Consortium effort, 10%> of the Japanese SNP database and 50%> of the HGBASE SNP database.
  • the data in dbSNP is integrated with other NCBI genomic data.
  • the output from the search is a dbSNP accession number, which is then tied in silico to identification and characterization of genomic landscape features including known genes, predicted genes, functional location and physical location in the genome.
  • Functional location specifies where the SNP falls within a gene or predicted gene, and details the location as exonic, promotor, intronic, 5' and 3' untranslated flanking region.
  • the physcial location includes the base pair position of the SNP on the individual chromosome. The base pairs that make up a chromosome are counted from the p telomere to the q telomere, starting with the first base pair on the p telomere.
  • the physical location also includes the cytoband designation that contains the SNP of interest.
  • the dbSNP search returns an accession # with an RS designation. This designation indicates that the SNP is a unique SNP identified as common between multiple studies.
  • the RS designation is used to perform additional database mining to harvest information relating to allele frequencies, penetrance estimates and heterozyosity estimates.
  • LocusLink provides a single query interface to curated sequence and descriptive information about genetic loci. It presents information on official nomenclature, aliases, sequence accessions, phenotypes, EC numbers, MIM numbers, UniGene clusters, homology, map locations, protein domains, and related web sites.
  • LocusLink accession number (LocusID), an NCBI genomic contig number (NT#), a reference mRNA number (NM#), splice site variants of the reference mRNA (XM#), a reference protein number (NP#), an OMIM accession number, and a Unigene accession number (HS#).
  • iii. Disease Association Databases Following the LocusLink search, the information returned is used to search disease association databases.
  • the HUGO Mutation Database Initiative which contains a collection of links to SNP/mutation databases for specific diseases or genes, is searched.
  • the OMIM database is searched. OMIM (Online Mendelian
  • Output from OMLM includes a modified accession number where multiple SNPs are associated with a genetic disorder. The number is annotated to designate the presence of multiple SNPs associated with the genetic disorder.
  • UniGene software (e.g., including but not limited to, UniGene) is used to partition search results into gene-oriented clusters.
  • UniGene is a system for automatically partitioning GenBank sequences into a non-redundant set of gene- oriented clusters.
  • Each UniGene cluster contains sequences that represent a unique gene, as well as related information such as the tissue types in which the gene has been expressed and map location.
  • EST novel expressed sequence tag
  • Unigene can be searched using either the UniGene accession number identified using LocusLink (prefened if available) or can be BLAST searched using the SNP target sequence of interest in FASTA format.
  • masked sequences are used to search the SNP Consortium (TSC) database (available at SNP Consortium Ltd public web site).
  • SNP Consortium searches are conducted concunently with dbSNP, LocusLink, UniGene, and OMIM searches.
  • the SNP Consortium database includes mapping and allele frequency information.
  • the database is searched via BLAST using the masked input target sequence.
  • the output from the SNP Consortium database includes a TSC accession number and a Goldenpath Contig accession number in addition to mapping and allele frequency information (if known).
  • target sequences are used to search genome databases (e.g., including but not limited to the Golden Path Database at University of California at Santa Cruz (UCSC) and GenBank).
  • the GoldenPath database is searched via BLAST using the sequence in FASTA format or using the RS# obtained from dbSNP.
  • GenBank is searched via BLAST using the masked sequence in FASTA format.
  • GoldenPath and GenBank searches are performed concurrently with TSC and dbSNP searches.
  • the searches result in the identification of the conesponding gene.
  • Output from GenBank includes a GenBank accession number.
  • Output from both databases includes contig accession numbers.
  • a match to an incomplete gene is identified.
  • the automated system of the present invention directs the search of databases of unfinished genomic sequences (e.g., including but not limited to The High Throughput Genomic (HTG) Sequences database, a database that includes unfinished sequences from DDBJ, EMBL, and GenBank).
  • Unfinished HTG sequences containing contigs greater than 2 kb are assigned an accession number and deposited in the HTG division.
  • a typical HTG record might consist of all the first pass sequence data generated from a single cosmid, BAC, YAC, or PI clone that together comprise more than 2 kb and contain one or more gaps.
  • accession number is assigned to this collection of sequences and each record includes a clear indication of the status (phase 1 or 2) plus a prominent warning that the sequence data is "unfinished” and may contain enors.
  • the accession number does not change as sequence records are updated; only the most recent version of a HTG record remains in GenBank. 'Finished' HTG sequences (phase 3) retain the same accession number, but are moved into the relevant primary GenBank division.
  • the information is transfened to the Oracle database of the present invention. If a gene is not identified, the automated system periodically (e.g., weekly) searches the databases for such information. vii. Private Databases
  • private databases are searched.
  • the present invention provides systems and methods for gathering, organizing, and storing sequence information (See e.g., Sections III, IV and V, below).
  • Information obtained by the methods of the present invention may be searched during target sequence analysis to assist in the confirmation or selection of target sequences that are likely to be successful in the desired detection assay (e.g., information obtained from previously successful assays is used to select or predict successful sequences for subsequent assays on the same or similar targets using the same or similar types of detection assay).
  • patent databases are searched.
  • a search is conducted to identify patents and patent applications related to a target or probe sequence.
  • patent claims may relate to target sequences, target SNPs, probe sequences and methods of using these compositions.
  • Searchable databases of patented sequences may be public or private. Examples of tools for searching for patented sequences include GENESEQ and The Patent Agent.
  • GENESEQ (Derwent Information, Alexandria VA) searches for patented sequences in basic patents from 40 patent issuing authorities worldwide.
  • GENESEQ provides a flat file (ASCII) EMBL-based format to enable integration into bioinformatics systems.
  • the Patent Agent DoubleTwist, Inc., Oakland, CA uses the
  • the collection of information obtained from the database searches is analyzed and/or stored.
  • the candidate target sequence is identified as a "high probability" target sequences and the results are reported (e.g. via the world wide web) to a user (to recommend production or use) or the target is directly sent on for production (Section III, below) or used.
  • a high probability target sequence is one where the target sequence was confirmed to exist in one or more sequence databases, where there is no identified disagreement between the sequence databases (e.g., disagreement relating to the sequence of the target, the location of the target, or the presence of known mutations within the target region), where the target sequence represents a unique sequence in the samples that are to be assayed, and where the sequence conesponding to the target is considered reliable (i.e., confirmed or completed) sequence.
  • the report may include results of each search, a summary of the results, a general indication that the target sequence is a high probability sequences, and/or any other detailed information identified by the searches (e.g., disease association information).
  • a report is sent (e.g. by the internet) to a user (e.g., the person who input or requested the candidate target sequence or a technician utilizing the systems and methods of the present invention) highlighting the one or more problems.
  • Problems include the presence of repeat or artifact sequences in the candidate target sequences, multiple copies of the target sequence in the sample to be assayed (e.g., in the human genome), absence of the sequence in one or more of the databases, inconsistent results from one or more the databases (e.g., inconsistency as to the sequence conesponding to the target, the location of the target within a genome, the presence or location of a mutation or SNP to be assayed, and the presence or absence of one or more additional mutations or SNPs within the target region), and or the sequence quality (reliability) of the sequence from the databases.
  • a reliability score is generated based on the presence or absence of one or more of the above potential problems.
  • the reliability score may be sent to the user, or may be used as a signal to cause a further action, such as to begin production and/or to cancel the candidate target sequence.
  • the user is given the option to select another target sequence or to proceed with the present target sequence (e.g., to proceed to production).
  • the systems of the present invention automatically select and test additional candidate target sequences based on the original requested candidate target sequence (e.g., select neighboring sequences and or remove problem portions of the sequence). If more reliable sequences are identified, these suggested alternate target sequences are reported to the user.
  • An overview of in silico analysis in some prefened embodiments of the present invention is shown in Figure 3.
  • the three top boxes represent exemplary sources of target sequences: research & development (e.g., direct input by research personnel) (20), Web interface (sequence , input through a communication network) (21), and system administrators (e.g., to test the systems and methods of the present invention) (22).
  • the target sequences are then analyzed by a screening component (23) that masks repeat and artifact sequences. If sequences are suitable for further analysis, they are passed to a series of databases. In the example shown in Figure 3, the sequences are simultaneously sent to dbSNP (24), GoldenPath (25), and SNP Consortium (26) databases.
  • dbSNP data (27) is collected and stored and the dbSNP accession number is used to search the Unigene database (29).
  • the dbSNP accession number may also be used to search the OMIM database (28) (which may also be searched after any other database search).
  • the target sequence information is passed to the Unigene database (29).
  • Unigene identification is found, Unigene data (30) is collected and stored.
  • the target sequence information sent to the GoldenPath database (25) is used to identify the base pair position of the SNP on the current GoldenPath assembly of the genome and to check the reliability status of the sequence.
  • sequence is considered “finished” sequence
  • GoldenPath data is collected and stored. If the sequence is not finished, the GenBank database (31) is searched to identify a GenBank contig identification number and to determine if the contig is considered “finished.” If the contig is finished, data is collected and stored. If the contig is not considered finished, a request for additional sequence data is placed with the group responsible with finishing the sequence of the region (32). If sequence data is available, data from the finishing group is collected and stored. The base pair position of the SNP generates the next level of in silico analysis to generate the genomic landscape information for each SNP resulting in a detailed in silico annotation of the SNP. The annotation is extended to include the full target sequence information.
  • Target sequences which fall within a known gene region defined as "genie" to include 10 kilobases of sequence 5' and 3' of the beginning and end of transcription, then a second round of in silico annotation charaterizes this genie region as well.
  • the target sequence information sent to the SNP Consortium database (26) is used to identify a TSC identification number and TSC data, if available, is collected and stored.
  • one or more database accession numbers e.g., LocusLink accession number
  • said accession numbers are used to direct searches in the conesponding database (e.g., LocusLink database) or other databases.
  • the panels are designed to map genes, to characterize novel mutations, to create disease-specific gene expression snapshots, to detect clinically relevant mutations, and to facilitate and direct clinical trials of novel treatments for disease. Allele frequency information is generated for each SNP and provides integration between each SNP and the published genetic and physical maps, as well as test algorithms for the prediction of the functional impact of amino acid changes in cSNPs.
  • the in silico analysis systems and methods described above allow the rapid development of products such as Analyte-Specific Reagents and In- vitro Diagnostics. Since the in silico analysis integrates sequence and expression data with literature and climcal data (e.g. data is fed back into the data management systems of the present invention) the product development funnel (See, section B.IV) if further promoted (See, Figure 5).
  • gene expression (GE) assays can be designed to ntimerous sites (e.g., from about 100 to several 1000 different sites) in a particular mRNA sequence. Further complicating the design process is determimng whether there is any homology between the RNA sequence of interest and any others that may be or are likely to be present in the sample. Homologies between target RNA and non-target RNAs occur not only in closely related gene families, but also when RNAs such as mRNAs have several alternative splice configurations.
  • the assay is intended to detect all or most members of a set of homologous DNAs or RNAs. In other embodiments, an assay is intended to detect a particular nucleic acid and to avoid detecting any similar or related sequences present in a sample. If significant homologies exist, sequence alignments performed before the assay is designed can identify sequences unique to a particular target from sequences that are shared. SNP variations that occur in the mRNA also need to be considered, as their position in the target region can affect assay performance, and location at or near the probe cleavage site may preclude detection of that particular variant. In some embodiments, this is a prefened effect; in some embodiments it is desirable to avoid this effect.
  • RNA target sequence i) splice sites, ii) accessible sites, and iii) discrimination sites.
  • the type of bioinformatic analysis performed on a given RNA target sequence depends on the type of design strategy being used for developing the assay.
  • Bioinformatic analysis in mRNA target sequence selection may include mapping of splice sites within the mRNA sequence, identification of any variations in the mRNA sequence (e.g. single-base changes, insertions, deletions), identification and alignment of splice variants, identification and alignment of closely related genes, homology to and alignment of the conesponding gene in other species, and location of accessible sites (unstructured regions of RNA) via in silico analysis.
  • sequences are obtained from and compared to information from a public database.
  • sequences are obtained from a private database and compared to information from a private and/or public database
  • relevant sequences are collected into a local database for rapid retrieval.
  • a fully integrated bioinformatic module includes complete analysis of the RNA target sequence prior to assay design, independent of how the assay will be designed. For example, in some embodiments, the user enters a GenBank NM_ accession number and the module retrieves the sequence, compares it to an mRNA sequence database (e.g., using BLAST) to retrieve sequences having a percent identity selected by the user (e.g., a minimum identity of 90%), aligns the target sequence with the retrieved sequences, and then uses subroutines to output positions where there is discrimination (e.g., 2 adjacent nucleotides) compared to the collection of retrieved sequences.
  • an mRNA sequence database e.g., using BLAST
  • additional subroutines comprise locating completely homologous regions of sequence relative to the collection of retrieved sequences for the design of inclusive assays (e.g., assays designed to detect all members of the collection).
  • subroutines are implemented that retrieve all known alternatively spliced variants, align them, and output splice junctions and included exons for the design of assays that either inclusively or exclusively detect these variants.
  • a subroutine performs a BLAST comparison of the mRNA sequence from one species against other databases for other species.
  • the output of the bioinformatics module comprises identification of splice sites for each RNA.
  • homologies are identified and used to design inclusive (e.g., interspecies) assays
  • inclusive e.g., interspecies
  • single assays can detect human and rat CYPl Al, or mouse and rat GAPDH, etc.
  • Interspecies assays have the benefits of making product development more efficient and less expensive, since two or more assays are developed, packaged, and inventoried for the time and price of one.
  • homologies are identified and used to design exclusive assays (e.g., assays that will not cross-react between species).
  • the output of a bioinformatics module is exported to an INVADERCREATOR module.
  • the information is manually entered into the INVADERCREATOR software, while in other embodiments it is read in, e.g., via a batch file.
  • batch files comprise numerical locations for sequences selected as targets for assay design.
  • other relevant information for assay design such as full gene names, gene name abbreviations, locations of SNP variations are included in the batch files for direct import into INVADERCREATOR software.
  • the user selects a design method after reviewing the contents of the bioinformatics output file.
  • a pre-selected or default design method based on the content of the output file is automatically selected.
  • the bioinformatics module exports data having particular information regarding homologous sequences found, e.g., a threshold percentage identity value, and this output information directs the INNADERCREATOR module to default to a discrimination sites design method.
  • information is cross- referenced in the I ⁇ VADERLOCATOR software.
  • output from an INVADERCREATOR analysis is fed back into the bioinformatics module for further analysis.
  • the bioinformatics module verifies a design feature, e.g., verifies that the final design selection(s) have the intended inclusivity or exclusivity.
  • a target selected based on one set of criteria e.g., exclusivity within the RNAs of a single species
  • a database using different criteria e.g. , cross-species homologies.
  • the output of the second analysis in the bioinformatics module is returned to the INVADERCREATOR module and the user is offered the option of altering an aspect of the assay design.
  • alteration or refinement of the assay design is an automated step based on the output from the informatics analysis.
  • inventoried assay sequences are reviewed against newly updated databases.
  • users are notified of new information (e.g., via
  • INVADERLOCATOR software related to previously characterized target sequences, such as newly identified SNPs or splice variants.
  • Detection Assay Design There are a wide variety of detection technologies available for determining the sequence of a target nucleic acid at one or more locations. For example, there are numerous technologies available for detecting the presence or absence of SNPs. Many of these techniques require the use of an oligonucleotide to hybridize to the target. Depending on the assay used, the oligonucleotide is then cleaved, elongated, ligated, disassociated, or otherwise altered, wherein its behavior in the assay is monitored as a means for characterizing the sequence of the target nucleic acid. A number of these technologies are described in detail, in Section V, below.
  • the present invention provides systems and methods for the design of oligonucleotides for use in detection assays.
  • the present invention provides systems and methods for the design of oligonucleotides that successfully hybridize to appropriate regions of target nucleic acids (e.g., regions of target nucleic acids that do not contain secondary structure) under the desired reaction conditions (e.g., temperature, buffer conditions, etc.) for the detection assay.
  • the systems and methods also allow for the design of multiple different oligonucleotides (e.g., oligonucleotides that hybridize to different portions of a target nucleic acid or that hybridize to two or more different target nucleic acids) that all function in the detection assay under the same or substantially the same reaction conditions.
  • These systems and methods may also be used to design control samples that work under the experimental reaction conditions.
  • the present invention also provides methods for designing sequences for amplifying the target sequence to be detected (e.g. designing PCR primers for multiplex PCR).
  • the INVADER assay provides means for forming a nucleic acid cleavage structure that is dependent upon the presence of a target nucleic acid and cleaving the nucleic acid cleavage structure so as to release distinctive cleavage products (See, Figure 6).
  • 5' nuclease activity for example, is used to cleave the target-dependent cleavage structure and the resulting cleavage products are indicative of the presence of specific target nucleic acid sequences in the sample.
  • invasive cleavage can occur.
  • the cleavage agent e.g., a 5' nuclease
  • the upstream oligonucleotide can be made to cleave the downstream oligonucleotide at an internal site in such a way that a distinctive fragment is produced.
  • the INVADER assay provides detections assays in which the target nucleic acid is reused or recycled during multiple rounds of hybridization with oligonucleotide probes and cleavage of the probes without the need to use temperature cycling (i.e., for periodic denaturation of target nucleic acid strands) or nucleic acid synthesis (i.e., for the polymerization-based displacement of target or probe nucleic acid strands).
  • temperature cycling i.e., for periodic denaturation of target nucleic acid strands
  • nucleic acid synthesis i.e., for the polymerization-based displacement of target or probe nucleic acid strands.
  • a cleavage reaction is run under conditions in which the probes are continuously replaced on the target strand (e.g. through probe- probe displacement or through an equilibrium between probe/target association and disassociation, or through a combination comprising these mechanisms, (Reynaldo, et al, J. Mol, Biol. 97: 511
  • the INVADER assay may also employ degenerate oligonucleotides (e.g. degenerate INVADER and probe oligonucleotides).
  • degenerate INVADER and probe oligonucleotides may be randomly changed at one more positions such that a set of degenerate INVADER and/or probe oligonucleotides are produced.
  • Degenerate sets of INVADER and probe oligonucleotides are particularly useful for use in conjunction with target sequences that tend to be heavily mutated (e.g. HIV-1 pol gene). Using such degenerate sets of INVADER and probe oligonucleotides allows the presence of target sequences at a particular location to be detected even if the sunounding sequence no longer represent the wild type or expected sequence.
  • the INVADER assay technology may be used to quantitate mRNA (e.g. without target amplification).
  • Low variability (3-10 % coefficient of variation) provides accurate quantitation of less than two-fold changes in mRNA levels.
  • a biplex FRET-based detection format enables simultaneous quantitation of expression from two genes within the same sample.
  • One of these genes can be an invariant housekeeping gene that is used as the internal standard. Normalizing the signals from the gene of interest with the internal standard provides accurate results and obviates the need for replicate samples.
  • a simple and rapid cell lysate sample preparation method can be used with the mRNA INVADER Assay. The combined features of biplex detection and easy sample preparation make this assay readily adaptable for use in high- throughput applications.
  • the INVADER assay (and other detection assays such as TAQMAN) employ an E-TAG label (e.g. as part of the INVADER oligonucleotide, probe oligonucleotide, or the FRET oligonucleotide).
  • E-TAG labeling is particularly useful in muliplex analysis. E-TAG labeling does not require surface immobilization of affinity agents. E-TAG type labeling is described in U.S. Pat.
  • oligonucleotide Design for the INVADER assay The application of the INVADER assay is not limited to any particular type of nucleic acid or nucleic acid variations.
  • oligonucleotides for an INVADER assay are designed to detect a particular SNP.
  • the oligonucleotides for an assay may be designed to determine the presence or absence of a particular nucleic acid in a sample, e.g., a nucleic acid suspected to be present as a consequence of, for example, transfection, transformation or infection of the source of the sample.
  • the oligonucleotides of an INVADER assay may be designed to provide quantitative information about a particular DNA or RNA sequence
  • sequences of interest are entered into the INVADERCREATOR program (Third Wave Technologies, Madison, WI).
  • INVADERCREATOR program Tin Wave Technologies, Madison, WI.
  • sequences may be input for analysis from any number of sources, either directly into the computer hosting the INVADERCREATOR program, or via a remote computer linked through a communication network (e.g., a LAN, Intranet or Internet network).
  • the program For detection of double-stranded nucleic acid, e.g., a gene, the program designs probes for both strands, e.g., the sense and antisense strands. Selection of a particular strand for detection is generally based upon factors that include the ease of synthesis, minimization of secondary structure formation, manufacturability 'and ⁇ NVADERCREATOR penalty scores, which have been established by studying probe design performance in the INVADER assay. In some embodiments, the user chooses the strand for sequences to be designed for. In other embodiments, the software automatically selects the strand.
  • oligonucleotide probes may be designed to operate at a pre-selected assay temperature (e.g., 63°C).
  • a final probe set (e.g., primary probes for 2 alleles and an INVADER oligonucleotide for a SNP detection assay, or primary probe, a stacker oligonucleotide, an INVADER oligonucleotide and an ARRESTOR oligonucleotide for an RNA detection assay) is selected.
  • the INVADERCREATOR system is a web-based program with secure site access that contains a link to BLAST (available at the National Center for
  • RNAstructure can test the proposed oligonucleotide designs generated by INVADERCREATOR for potential uni- and bimolecular complex formation.
  • INVADERCREATOR is open database connectivity (ODBC)-compliant and uses the Oracle database for export/integration.
  • ODBC open database connectivity
  • the INVADERCREATOR system is configured with ORACLE to work well with UNIX systems, as most genome centers are UNIX-based.
  • the INVADERCREATOR analysis is provided on a separate server (e.g., a Sun server) so it can handle analysis of large batch jobs. For example, a customer can submit up to 2,000 SNP sequences in one email.
  • the server passes the batch of sequences on to the INVADERCREATOR software, and, when initiated, the program designs detection assay oligonucleotide sets.
  • probe set designs are returned to the user within 24 hours of receipt of the sequences.
  • Each INVADER reaction includes at least two target sequence-specific, unlabeled oligonucleotides for the primary reaction: an upstream INVADER oligonucleotide and a downstream Probe oligonucleotide.
  • the INVADER oligonucleotide is generally designed to bind stably at the reaction temperature, while the probe is designed to freely associate and disassociate with the target strand, with cleavage occurring only when an uncut probe hybridizes adjacent to an overlapping INVADER oligonucleotide.
  • the probe includes a 5' flap or "arm" that is not complementary to the target, and this flap is released from the probe when cleavage occurs.
  • the released flap participates as an INVADER oligonucleotide in a secondary reaction.
  • the INVADER reaction may comprise additional oligonucleotides, such as stacker or ARRESTOR oligonucleotides.
  • the designed oligonucleotides are submitted as a synthesis order, such that manufacture of each oligonucleotide is initiated at order submission, are tracked through the modules of synthesis and the manufactured set of oligonucleotides are collected into a finished assay product or kit.
  • the oligonucleotide designs are checked against an inventory of existing oligonucleotides to determine if any of the oligonucleotides of the assay have been previously synthesized ("pre-synthesized" oligonucleotides) and stored.
  • one or more pre-synthesized oligonucleotides are taken from inventory oligonucleotides and included with newly designed and synthesized oligonucleotides in the finished assay or kit.
  • new assays or kits are assembled entirely from pre-synthesized oligonucleotides taken from an inventory of oligonucleotides.
  • an INVADERCREATOR program is configured to design oligonucleotides for an assay of a single particular type or pmpose (e.g., for SNP detection or RNA quantitation).
  • an INVADERCREATOR program is configured to allow a user to select, e.g., through a button, check box or menu, from a variety of assay types or pwposes. The following discussion provides several examples of how a user interface for an INVADERCREATOR program may be configured. Examples of user interfaces are presented in Figures 12 through 14.
  • Figure 12 provides screens images showing one example of using an INVADERCREATOR program to designs an assay for the detection of a SNP (a SNP INVADERCREATOR, or SIC program module).
  • Figure 13 provides a selection of screen images showing one example of using an INVADERCREATOR program to design an assay for the detection of an RNA target (an RNA INVADERCREATOR, or RIC program module).
  • Figure 14 provides a selection of screen images showing one example of using an INVADERCREATOR program to design an assay for the detection of a transgene (a Transgene INVADERCREATOR, or TIC program module) .
  • screens provide optional selection of any number of modifications (e.g., arms, dyes, detectable moieties) for detection or further manipulation.
  • an INVADERCREATOR module may be customized for a particular assay, or for the needs of a particular user or customer. For example, if a customer has a particular detection platform requiring that the cleavage products comprise moiety X, an INVADERCREATOR module can be configured such that all assays designed by or for customer X are automatically configured to comprise moiety X, in accordance with the customer's requirements.
  • a pre-designated design feature cannot be altered by an operator creating a new probe design using the customized INVADERCREATOR module.
  • a pre- designated design feature may be presented to an operator as a default condition of the design that may be overridden during probe design (e.g., by selecting an alternative configuration through one or more data entry screens).
  • an INVADERCREATOR program the user initiates oligonucleotide design by opening a work screen (e.g., Figures 12A, 13 A or 14A), e.g., by clicking on an icon on a desktop display of a computer (e.g., a Windows desktop).
  • a work screen e.g., Figures 12A, 13 A or 14A
  • the user enters information related to the assay, such as project code, company name, assay name, etc.
  • the used indicates what species the nucleic acid sequence is from.
  • the user selects the INVADERCREATOR program module to be used (e.g., SIC, RIC, TIC, etc.), e.g., by clicking a button on the screen.
  • the user enters information related to the target sequence for wliich an assay is to be designed.
  • the user enters a target sequence (e.g., Figures 12B, 13C, or 14B).
  • the user enters a code or number that causes retrieval of a sequence from a database.
  • additional information may be provided, such as the user's name, an identifying number associated with a target sequence, and/or an order number.
  • the user indicates (e.g. via a check box or drop down menu) that the target nucleic acid is DNA or RNA.
  • the user indicates the species from which the nucleic acid is derived.
  • the user indicates whether the design is for monoplex (i.e., one target sequence or allele per reaction) or multiplex (i.e., multiple target sequences or alleles per reaction) detection.
  • the user starts the analysis process, fri one embodiment, the user clicks a "Design It" button to continue.
  • the software validates the field entries before proceeding. In some embodiments, the software verifies that any required fields are completed with the appropriate type of information. In other embodiments, the software verifies that the input sequence meets selected requirements (e.g., minimum or maximum length, DNA or RNA content). If entries in any field are not found to be valid, an enor message or dialog box may appear. In prefened embodiments, the enor message indicates which field is incomplete and/or inconect. Once a sequence entry is verified, the software proceeds with the assay design.
  • selected requirements e.g., minimum or maximum length, DNA or RNA content
  • the information supplied in the order entry fields specifies what type of design will be created.
  • the target sequence and multiplex check box specify which type of design to create. Design options include but are not limited to SNP assay, Multiplexed SNP assay (e.g., wherein probe sets for different alleles are to be combined in a single reaction), Multiple SNP assay (e.g., wherein an input sequence has multiple sites of variation for which probe sets are to be designed), and Multiple Probe Arm assays.
  • the INVADERCREATOR software is started via a Web Order
  • WebOE WebOE Entry
  • the user chooses two or more designs to work with. In some embodiments, this selection opens a new screen view (e.g., a Multiple SNP Design Selection view Figure 8).
  • the software creates designs for each locus specified in the target sequence, scoring each, and presents them to the user in this screen view. The user can then choose any two designs to work with. In some embodiments, the user chooses a first and second design (e.g., via a menu or buttons) and clicks a "Design It" button to continue.
  • the melting temperature (T m ) of the SNP to be detected is calculated using the nearest-neighbor model and published parameters for DNA duplex formation (Allawi and SantaLucia, Biochemistry, 36:10581 [1997], SantaLucia, Proc Natl Acad Sci U S A., 95(4):1460 . [1998]).
  • T m melting temperature
  • RNA/DNA heteroduplex formation may be used. Because the assay's salt concentrations are often different than the solution conditions in which the nearest-neighbor parameters were obtained (1M NaCl and no divalent metals), an adjustment should be made to the value provided for the salt concentration within the melting temperature calculations. This adjustment is termed a 'salt conection' SantaLucia, Proc Natl Acad Sci U S A., 95(4):1460 [1998]. Similarly, the presence and concentration of the enzyme influence optimal reaction temperature. One way of compensating for these additional factors is to further vary the salt value in the Tm calculations.
  • salt conection refers to a variation made in the value provided for a salt concentration for the pu ⁇ ose of reflecting the effect on a T m calculation for a nucleic acid duplex of a both an alternative salt effect and a non-salt parameter or condition affecting said duplex. Variation of the values provided for the strand concentrations will also affect the outcome of these calculations.
  • the algorithm used for calculating probe-target melting temperature has been adapted for use in predicting optimal INVADER assay reaction temperatures. For one set of 30 probes, the average deviation between optimal assay temperatures calculated by this method and those experimentally determined is about 1.5 °C.
  • the length of the target-complementary region of a probe is defined by the temperature selected for running the reaction (e.g., 63°C). Starting from the target base that is paired to the probe nucleotide 5' of the intended cleavage site (e.g. , the position of the variant nucleotide on the target DNA)), and adding on the 3' end, an iterative procedure is used by which the length of the target-binding region of the probe is increased by one base pair at a time until a calculated optimal reaction temperature (T m plus salt conection to compensate for enzyme effect) matching the desired reaction temperature is reached.
  • T m plus salt conection to compensate for enzyme effect a calculated optimal reaction temperature matching the desired reaction temperature
  • the non-complementary arm of the probe is preferably selected to allow the secondary reaction to cycle at the same reaction temperature.
  • the entire probe oligonucleotide is screened using programs such as mfold (Zuker, Science, 244: 48 [1989]) or Oligo 5.0 (Rychlik and Rhoads, Nucleic Acids Res, 17: 8543 [1989]) for the possible formation of dimer complexes or secondary structures that could interfere with the reaction.
  • mfold Zauker, Science, 244: 48 [1989]
  • Oligo 5.0 Oligo 5.0
  • the stability of the INVADER oligonucleo tide-target hybrid exceeds that of the probe (and therefore the planned assay reaction temperature), generally by 15-20 °C.
  • the 3' end of the INVADER oligonucleotide is designed to have a nucleotide not complementary to either allele suspected of being contained in the sample to be tested. The mismatch does not adversely affect cleavage (Lyamichev et al, Nature Biotechnology, 17: 292 [1999]), and it can enhance probe cycling, presumably by mimmizing coaxial stabilization effects between the two probes.
  • all of the probe sequences may be selected to allow the primary and secondary reactions to occur at the same optimal temperature, so that the reaction steps can run simultaneously.
  • the probes may be designed to operate at different optimal temperatures, so that the reaction steps are not simultaneously at their temperature optima.
  • the software provides the user an opportunity to change various aspects of the design including but not limited to: probe, target and INVADER oligonucleotide temperature optima and concentrations; blocking groups; probe arms; dyes, capping groups and other adducts; individual bases of the probes and targets (e.g., adding or deleting bases from the end of targets and/or probes, or changing internal bases in the INVADER and/or probe and/or target oligonucleotides).
  • changes are made by selection from a menu.
  • changes are entered into text or dialog boxes. In prefened embodiments, this option opens a new screen (e.g., a Designer Worksheet view, Figure 9).
  • the software provides a scoring system to indicate the quality (e.g., the likelihood of performance) of the assay designs.
  • the scoring system includes a starting score of points (e.g., 100 points) wherein the starting score is ' indicative of an ideal design, and wherein design features known or suspected to have an adverse affect on assay performance are assigned penalty values. Penalty values may vary depending on assay parameters other than the sequences, including but not limited to the type of assay for which the design is intended (e.g., DNA, RNA, monoplex, multiplex) and the temperature at which the assay reaction will be performed. The following example provides illustrative scoring criteria for use with some embodiments of the INVADER assay based on an intelligence defined by experimentation.
  • Examples of design features in assays for DNA detection that may incur score penalties include but are not limited to the following [penalty values are indicated in brackets; if there are 2 numbers, the first number is for lower temperature assays (e.g., 62-64 °C), second is for higher temperature assays (e.g., 65-66 °C)]:
  • Arm 2 ATGACGTGGCAGAC 5' AGACX or 5' AGACXX
  • Arm 3 ACGGACGCGGAG 5' GGAGX or 5' GGAGXX
  • probe has a 5-base stretch containing the polymo ⁇ hism
  • probe has a 5-base stretch adjacent to the polymo ⁇ hism
  • probe has a 4-base stretch of Gs containing the polymo ⁇ hism
  • probe has a 5-base stretch of Gs - penalty added anytime it is infringed 7.
  • INVADER oligonucleotide 6-base stretch is of Gs - additional penalty 8.
  • two or three base sequence repeats at least four times starting in the region +1 to +4 of the probe.
  • probe hybridizing region is short ⁇ 12 bases regardless of assay temperature.
  • probe hybridizing region is long (> 26 bases).
  • a probe has a calculated Tm 2C less than its target Tm
  • RNA detection e.g., RIC module penalties
  • score penalties include but are not limited to the following:
  • probe has 4-G stretch in the INVADER oligonucleotide, probe, or stacker.
  • probe has 5-base stretch containing position 1
  • probe has 5-base stretch containing position 2
  • probe hybridizing region is short (8 bases with a stacker or ⁇ 12 bases without a stacker)
  • probe hybridizing region is long (> 17 bases with a stacker or > 20 bases without a stacker)
  • penalties are assessed for location of SNP variations at or near the cleavage site. In other embodiments, penalties are assessed based on cleavage site base preferences (e.g., some enzyme may cleave after more efficiently after particular bases, such as Gs, and penalties may be used when a different base is placed in that location). In still other embodiments, penalties are assessed based on ranking of stacking interactions between a probe 3' base and a stacking oligonucleotide 5' base (e.g., in some embodiments, AA stacks may perform better than TT stacks.
  • temperatures for each of the oligonucleotides in the designs are recomputed and scores are recomputed as changes are made.
  • score descriptions can be seen by clicking a "descriptions" button.
  • a BLAST search option is provided.
  • a BLAST search is done by clicking a "BLAST Design” button. In some embodiments, this action brings up a dialog box describing the BLAST process.
  • the BLAST search results are displayed as a highlighted design on a Designer Worksheet.
  • a user accepts a design by clicking an "Accept" button.
  • the program approves a design without user intervention.
  • the program sends the approved design to a next process step (e.g., into production; into a file or database).
  • the program provides a screen view (e.g., an Output Page, Figure 10 OLD NUMBER), allowing review of the final designs created and allowing notes to be attached to the design.
  • the user can return to the Designer Worksheet (e.g., by clicking a "Go Back” button) or can save the design (e.g., by clicking a "Save It” button) and continue (e.g., to submit the designed oligonucleotides for production).
  • the program provides an option to create a screen view of a design optimized for printing (e.g., a text-only view) or other export (e.g., an Output view, Figure 11).
  • the Output view provides a description of the design particularly suitable for printing, or for exporting into another application (e.g., by copying and pasting into another application).
  • the Output view opens in a separate window.
  • a design session using the RIC module for RNA assay design is represented in Figure 13.
  • the RIC module is shown by way of example; similar steps are followed in the SIC and TIC design modules represented in Figures 12 and 14, respectively.
  • RNA assay design in this embodiment of the RIC module may comprise the following steps:: • entry of assay information into defined fields (e.g., user, assay name, assay abbreviation, etc.) (Figure 13A). » user selects species via drop down menu ( Figure 13B).
  • defined fields e.g., user, assay name, assay abbreviation, etc.
  • RNA sequences (including FASTA format) is copied and pasted in ( Figure 13C).
  • cleavage site based design is indicated (e.g., sites indicated are splice junctions, SNPs, or other any other sites selected by user, for example, using the bioinformatics assessment described above; user can enter multiple sites) ( Figure 13C).
  • Multiple probes can be designed per cleavage site (e.g., 257[3] gives three probes for the design for the 257 site).
  • Stacking oligonucleotide design format can be selected (e.g., "Has Stacker” button, Figure 13C).
  • Bases can be added to or deleted from the 5' end of the INVADER oligonucleotide( Figure 13E), the 3' end of the probe (automatically adjusts stacking oligonucleotide position and length to satisfy it temperature setting) ( Figure 13F), and the
  • the user can alter the INVADER oligonucleotide, probe, and stacking oligonucleotide temperatures (e.g., Fig 13G). Exemplary default settings and actual calculated values are shown (e.g., in a separate window). .
  • the user can alter the target, INVADER oligonucleotide, probe, and stacking oligonucleotide concentrations e.g., from default settings( Figure 13H); .
  • user can select enzymes (e.g., alternative CLEAVASE enzymes) via drop-down menu. .
  • Design Review shows all entered assay information, the complete mRNA sequence (5' to 3'), and the designed INVADER oligonucleotide set for each cleavage site aligned to its conesponding mRNA sequence (displayed here 3' to 5')
  • the user gets a listing of all oligonucleotides that were checked for ordering in the Design Review screen and selects each one to call up the oligo order form for that particular oligonucleotide (Figure 13J), • An Oligo Request form is queued up for each oligo and the user has the ability to select an oligo type via a drop-down menu, the synthesis scale, purification method, various 5', 3', or internal modifications, the ability to select "Other” and input unique modifications not listed in the drop-down menus, the ability to highlight a portion of the sequence and designate and alternative nucleotide chemistry (e.g., 2'-Ome's or phosphorothioates) (13 L-0).
  • nucleotide chemistry e.g., 2'-Ome's or phosphorothioates
  • the software is set to automatically accept default values and submit all orders directly from the Design Review screen (e.g., via n "order Oligonucleotides Now” button) without user review of an Oligo Request form. .
  • the user selects the "Submit to Synthesis” button when finished modifying a particular Oligo Request form and then queues up the remaining oligonucleotides in the order one by one and does likewise.
  • the RIC module also allows the selection of multiple designs for one cleavage site. For example, entering "257, 257, 257, 512" in the sites box (e.g., on Figure 13C for 13P) would give the same three designs for 257 and one for 512. As shown in 13P, one could also enter 257 [2] to create 2 designs to the 257 site. In some embodiments, the user has the ability to modify each design individually in the following steps.
  • a redesign method could include moving the cleavage site/accessible site 1 or more nucleotides in either direction and/or lower scoring designs not ordered in the initial process could be ordered and tested.
  • Integration of the various design methods could involve querying the user or having the user select one or more design methods based on the following examples:
  • Splice site design involves determining the splice junctions within the mRNA, usually via pairwise alignment of the mRNA sequence with the genomic DNA sequence for that gene, and then locating INVADER assay cleavage sites at or near the splice site.
  • the INVADER oligonucleotide is positioned on one side of the splice junction and the probe and stacking oligonucleotide (if used) are positioned on the other side.
  • the probe and INVADER oligonucleotides would be separated by the intervening intronic sequences, which would preclude formation of the required overlap substrate for the CLEAVASE enzyme.
  • Accessible site design Again, if assay specificity and/or performance requirements do not dictate otherwise, assays can also be designed to accessible sites within the mRNA. Accessible sites are unstructured regions of the RNA and those determined experimentally, for example, using RT-ROL (AUawi et al RNA 7:314 [2001]), usually conelate well with enhanced INVADER RNA assay performance. Accessible sites can also be determined via in silico analysis. For example, the RNA sequence could be folded in m-Fold software and then analyzed in Oligowalk to determine accessible sites in the RNA. A program could be written to automatically output the accessible sites (defined as a region with negative Overall G values for an oligonucleotide binding to that region) for the folded RNA.
  • RT-ROL AUawi et al RNA 7:314 [2001]
  • the program could deteimine when there were 5 or more consecutive nucleotides with Overall G values of- 5 or less, then determine the midpoint of this region, and then output those sites into a file.
  • a 10-base negative G region encompassing target sequence nucleotides 200-210 would conespond to an accessible site at 205.
  • accessible site design could be encoded into the INVADERCREATOR module by method A or B .
  • Assays could be designed in reverse of the cleavage site design process. The user would specify the precise position of the 3' end of the probe within an accessible site and the probe would be biiilt out toward the 5' end to satisfy the preset Tm requirement. Stacking oligonucleotide (if designing in a stacker format) contributions to the probe's Tm would be determined as the probe was being built and the Invader oligonucleotide would be designed after the program finished the probe or probe/stacker design.
  • Hatim Allawi suggested an alternative method for accessible site design that could use the same probe-building algorithm that is used for cleavage site design methods.
  • the user could enter the accessible site and the INVADERCREATOR module could shift a defined number of bases (a default shift could be determined) downstream. For example, 200 could be entered as an accessible site, and INVADERCREATOR module would build a design using the existing algorithm for cleavage site 210 if the shift value was 10.
  • Next to the check box for "Stacker Design” could be a check box for "Accessible Site Design”.
  • Next to this check box could be a field in which the user would designate the number of bases to shift.
  • the cunent "Cleavage Sites” field could say “Design Sites” to genetically encompass either design mode (cleavage sites or accessible sites). Users could have the capability to check one or both boxes (e.g. stacker design and accessible site design, accessible site design only, etc.).
  • Splice variant design Splice variant assays can be designed in a variety of ways.
  • An inclusive detection assay could be designed to detect a region of sequence (e.g. a particular exon) present in all variants.
  • a particular splice variant could be detected by designing the assay to a unique splice site (e.g. if a 5 exon gene yields a splice variant that excludes exon 3, the assay could be designed to detect the exon 2-exon 4 splice junction). Since specificity of the INVADER RNA assay is primarily linked to discrimination at the cleavage site, even very small exonic sequences (e.g. a few nucleotides) could be distinguished.
  • the quantitation pattern from this type of INVADER RNA assay analysis may conelate with particular cellular processes or metabolic states.
  • Discrimination site design Closely-related sequences would be aligned to the input target sequence and an automated analysis could be performed to identify all sites that contain, for example, two or more adjacent base differences for any one sequence from all others in the alignment.
  • Another automated analysis algorithm could determine regions of homology of sufficient size to accommodate an INVADER oligonucleotide probe set that would inclusively detect all closely-related mRNAs. An output of the location of such double base discrimination sites or regions of homology could be reviewed by the user before accessing the INVADERCREATOR module or automatically designed via input of a batch file.
  • the present invention is not limited to the use of the INVADERCREATOR software. Indeed, a variety of software programs are contemplated and are commercially available, including, but not limited to GCG Wisconsin Package (Genetics computer Group, Madison, WI) and Vector NTI (Informax, Rockville, Maryland).
  • the present invention provides design parameters for combining multiple nucleic acid detection technologies.
  • INVADER assays or other assays are used in conjunction with amplified nucleic acid obtained by using the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • PCR is run simultaneously with other assays.
  • Probes The following are example of considerations that may be used when designing TAQMAN probes.
  • One consideration is to design PCR primers such that the amplicon size is between 50 - 150 base pairs.
  • Another consideration is to design PCR primers that have a Tm of around 60°C, with less than 2°C difference in Tm between forward and reverse primers.
  • Prefened primers have GC% around 40-60%> and have three or less consecutive runs of any nucleotide.
  • the primers Preferably, have total lengths of between 18-25 nucleotides in length.
  • PCR Primers are designed to have minimal haripin and minimal dimer formation tendencies (See below).
  • the TAQMAN probe is then chosen from within the amplicon region, and has a Tm of about 10°C higher than the Tm of the PCR primers (typically, 70°C).
  • TAQMAN probes should have a 5 ' FAM and a 3 ' TAMRA (or other labels), and not begin with G.
  • TAQMAN probes may be chosen, for example, by using programs such as Oligo Walk to scan through the amplicon sequence and a probe chosen based upon predicted most stable thermodynamic parameters.
  • candidate TAQMAN probes can be eliminated which forms more than three consecutive basepairs with the PCR primers.
  • the INVADER assay can be used for the detection of single nucleotide polymo ⁇ hisms (SNPs) with as little as 100-10 ng of genomic DNA without the need for target pre-amplification.
  • SNPs single nucleotide polymo ⁇ hisms
  • the amount of sample DNA becomes a limiting factor for large-scale analysis.
  • multiplex PCR coupled with the INVADER assay requires only limited target amplification (10 3 -10 4 ) as compared to typical multiplex PCR reactions that require extensive amplification (10 9 -10 12 ) for conventional gel detection methods.
  • the low level of target amplification used for INVADER assay detection provides for more extensive multiplexing by avoiding amplification inhibition commonly resulting from target accumulation.
  • the present invention provides methods to overcome such problems, by generating a unique target sequence using a nucleic acid amplification technique (e.g., PCR), such that the unique target sequence is tested by the detection assay, rather the original sample (e.g., genomic DNA).
  • a nucleic acid amplification technique e.g., PCR
  • This method is compatible with multiplexing, where considerations are made to ensure that amplified target sequence meets several criteria: 1) that the target sequence contains the polymo ⁇ hism to be analyzed; 2) that the target sequence represents a unique target sequence (i.e., it is the only sequence in the reaction mixture that is detected by a detection assay designed to target the target sequence); and 3) that the target sequence does not contain other polymo ⁇ hisms that are detected by any of the detection assays present in the multiplex reaction.
  • Suitable detection assay components may be selected with methods similar to those described above for the INVADERCREATOR methods.
  • the software performs a BLAST alignment of the target sequence used for the SNP assay to find similar sequences in the genome that may generate the cross-reactivity signal.
  • the design of PCR primers with software program should prevent amplification of any of the similar loci except the locus containing the SNP.
  • the software performs a BLAST alignment of the sequence amplified with a pair of primers against all other detection assay sequences included in the pool. If cross-reactivity or potential cross-reactivity exists, the set of primers is redesigned or the co-amplified sequences are included in different pools.
  • the same type of design analysis may be used for detection assays directed at the detection of haplotypes. For example, primers are generated to amplify sets of target sequences that each uniquely contain the polymo ⁇ hisms to be detected.
  • multiplex detection assays are provided in a plurality of arrays.
  • a first anay comprises assays configured for detection directly from genomic DNA and a second anay comprises assays configured for pre- amplification of target sequences from genomic DNA prior to detection assay analysis of the target sequence.
  • only limited pre-amplification of target sequences is carried out prior to detection by the detection assay. For example, in some embodiments, only a 10 5 -10 6 fold or less increase in target copy number is obtained prior to detection. This is in contrast to typical PCR reactions where 10 10 -10 12 or more fold amplification is utilized in detection reactions. In certain embodiments, 100 genotypes from a single PCR amplification are possible with the methods and systems of the present invention using only 10 ng of genomic DNA (e.g. less than 0.1 ng of human genomic DNA per SNP).
  • kits are provided for pre-amplification and detection of target sequences. In some embodiments, the kits comprise amplification primers.
  • the amplification primers may be provided in a single container.
  • the amplification primers may also be packaged with detection assay components.
  • amplification primers and detection assay components e.g., INVADER assay components
  • the reaction components are provided in dry form in a reaction chamber.
  • the kits are configured to allow reactions to occur where the only thing that is added to the reaction chamber is a solution containing genomic DNA.
  • the present invention provides methods and selection criteria that allow primer sets for multiplex PCR to be generated (e.g. that can be coupled with a detection assay, such as the INVADER assay).
  • software applications of the present invention automated multiplex PCR primer selection, thus allowing highly multiplexed PCR with the primers designed thereby.
  • MAP INVADER Medically Associated Panel
  • the methods, software, and selection criteria of the present invention allowed accurate genotyping of 94 of the 101 possible amplicons ( ⁇ 93%) from a single PCR reaction.
  • the original PCR reaction used only 10 ng of hgDNA as template, conesponding to less than 150 pg hgDNA per INVADER assay.
  • Figure 15 illustrates creation of one of the primer pairs (both a forward and reverse primer) for a 101 primer set from sequences available for analysis on the INVADER Medically Associated Panel using one embodiment of the software application of the present invention.
  • Figure 15A shows a sample input file of a single entry (e.g. shows target sequence information for a single target sequence containing a SNP that is processed the method and software of the present invention).
  • the target sequence information in Figure 15 includes Third Wave Technologies's SNP#, short name identifier, and sequence with the SNP location indicated in brackets.
  • Figure 15B shows the sample output file of a the same entry (e.g.
  • the output information includes the sequence of the footprint region (capital letters flanking SNP site, showing region where INVADER assay probes hybridize to this target sequence in order to detect the SNP in the target sequence), forward and reverse primer sequences (bold), and their conesponding Tm's.
  • the selection of primers to make a primer set capable of multiplex PCR is performed in automated fashion (e.g. by a software application). Automated primer selection for multiplex PCR may be accomplished employing a software program designed as shown by the flow chart in Figure 17.
  • the present invention provides methods and software application that provide selection criteria to generate a primer set configured for multiplex PCR, and subsequent use in a detection assay (e.g. INVADER detection assays).
  • a detection assay e.g. INVADER detection assays
  • the methods and software applications of the present invention start with user defined sequences and conesponding SNP locations.
  • the methods and/or software application determines a footprint region within the target sequence (the minimal amplicon required for INVADER detection) for each sequence (shown in capital letters in Figure 15B).
  • the footprint region includes the region where assay probes hybridize, as well as any user defined additional bases extending outward therefore (e.g. 5 additional bases included on each side of where the assay probes hybridize).
  • primers are designed outward from the footprint region and evaluated against several criteria, including the potential for primer-dimer formation with previously designed primers in the cunent multiplexing set (See, primers in bold in Figure 15 A, and selection steps in Figure 17).
  • This process may be continued, as shown in Figure 17, through multiple iterations of the same set of sequences until primers against all sequences in the cunent multiplexing set can be designed.
  • a primer set is designed for multiplex PCR, this set may be employed, in some embodiments,, as shown in the basic workflow scheme shown in Figure 16.
  • Multiplex PCR may be carried out, for example, under standard conditions using only 10 ng of hgDNA as template. After 10 min at 95°C, Taq (2.5 units) may be added to a 50ul reaction and PCR carried out for 50 cycles. The PCR reaction may be diluted and loaded directly onto an INVADER MAP plate (3ul/well) (See Figure 16).
  • the number of PCR reactions is from about 1 to about 10 reactions. In some embodiments, the number of PCR reactions is from about 10 to about 50 reactions. In further embodiments, the number of PCR reactions is from about 50 to about 100. In additional embodiments, the number of PCR reactions is greater than 100.
  • the present invention also provides methods to optimize multiplex PCR reactions (e.g. once a primer set is generated, the concentration of each primer or primer pair may be optimized). For example, once a primer set has been generated and used in a multiplex PCR at equal molar concentrations, the primers may be evaluated separately such that the optimum primer concentration is determined such that the multiplex primer set performs better.
  • Multiplex PCR reactions are being recognized in the scientific, research, clinical and biotechnology industries as potentially time effective and less expensive means of obtaining nucleic acid information compared to standard, monoplex PCR reactions. Instead of performing only a single amplification reaction per reaction vessel (tube or well of a multi-well plate for example), numerous amplification reactions are performed in a single reaction vessel.
  • the cost per target is theoretically lowered by eliminating technician time in assay set-up and data analysis, and by the substantial reagent savings (especially enzyme cost).
  • Another benefit of the multiplex approach is that far less target sample is required.
  • SNPs single nucleotide polymo ⁇ hisms
  • the concept of performing a single reaction, using one sample aliquot to obtain, for example, 100 results, versus using 100 sample aliquots to obtain the same data set is an attractive option.
  • primer dimers even if only a few bases in length, may inhibit both primers from conectly hybridizing to the target sequence. Further, if the dimers form at or near the 3' ends of the primers, no amplification or very low levels of amplification will occur, since the 3' end is required for the priming event. Clearly, the more primers utilized per multiplex reaction, the more abenant primer interactions are possible. The methods, systems and applications of the present help prevent primer dimers in large sets of primers, making the set suitable for highly multiplexed PCR.
  • primer pairs for numerous sites (for example 100 sites in a multiplex PCR reaction)
  • the order in which primer pairs are designed can influence the total number of compatible primer pairs for a reaction. For example, if a first set of primers is designed for a first target region that happens to be an A T rich target region, these primers will be A T rich. If the second target region chosen also happens to be an A T rich target region, it is far more likely that the primers designed for these two sets will be incompatible due to abenant interactions, such as primer dimers. If, however, the second target region chosen is not A/T rich, it is much more likely that a primer set can be designed that will not interact with the first A T rich set.
  • the present invention randomizes the order in which primer sets are designed (See, Figure 17). Furthermore, in some embodiments, the present invention re-orders the set of input target sequences in a plurality of different, random orders to maximize the number of compatible primer sets for any given multiplex reaction (See, Figure 17). In certain embodiments, the primers are designed such that GC-rich and AT-rich regions are avoided.
  • the present invention provides criteria for primer design that minimizes 3' interactions (e.g. 3' complementarity of primers is avoided to reduce probability of primer-dimer formation), while maximizing the number of compatible primer pairs for a given set of reaction targets in a multiplex design.
  • N[l] is an A or C (in alternative embodiments, N[l] is a G or T).
  • N[2]-N[l] of each of the forward and reverse primers designed should not be complementary to N[2]-N[l] of any other oligonucleotide.
  • N[3]-N[2]-N[l] should not be complementary to N[3]- N[2]-N[l] of any other oligonucleotide.
  • the next base in the 5 ' direction for the forward primer or the next base in the 3 ' direction for the reverse primer may be evaluated as an N[l] site. This process is repeated, in conjunction with the target randomization, until all criteria are met for all, or a large majority of, the targets sequences (e.g. 95%> of target sequences can have primer pairs made for the primer set that fulfill these criteria).
  • Another challenge to be overcome in a multiplex primer design is the balance between actual, required nucleotide sequence, sequence length, and the oligonucleotide melting temperature (Tm) constraints.
  • Tm oligonucleotide melting temperature
  • the primers in a multiplex primer set in a reaction should function under the same reaction conditions of buffer, salts and temperature, they need therefore to have substantially similar Tm's, regardless of GC or AT richness of the region of interest.
  • the present invention allows for primer design that meets minimum Tm and maximum Tm requirements and minimum and maximum length requirements.
  • x is selected such the primer has a predetermined melting temperature (e.g. bases are included in the primer until the primer has a calculated melting temperature of about 50 degrees Celsius).
  • each of the primers in a set has the same melting temperature.
  • the products of a PCR reaction are used as the target material for another nucleic acid detection means, such as a hybridization-type detection assays, or the INVADER reaction assays for example.
  • a hybridization-type detection assays or the INVADER reaction assays for example.
  • Selection criteria may be employed such that the primers designed for a multiplex primer set do not react (e.g. hybridize with, or trigger reactions) with oligonucleotide components of a detection assay.
  • certain homology criteria is employed.
  • each of the primers in the set are defined as 5'-N[x]-N[x-l]- -N[4]-N[3]-N[2]-
  • N[l]-3' then N[4]-N[3]-N[2]-N[l]-3' is selected such that it is less than 90% homologous with the FRET or INVADER oligonucleotides.
  • N[4]-N[3]-N[2]-N[l]-3' is selected for each primer such that it is less than 80% homologous with the FRET or INVADER oligonucleotides.
  • N[4]-N[3]-N[2]-N[l]-3' is selected for each primer such that it is less than 70%> homologous with the FRET or INVADER oligonucleotides.
  • Figure 17 shows a flow chart with the basic flow of certain embodiments of the methods and software application of the present invention.
  • the processes detailed in Figure 17 are inco ⁇ orated into a software application for ease of use (although, the methods may also be performed manually using, for example, Figure 17 as a guide).
  • Target sequences and or primer pairs are entered into the system shown in Figure 17.
  • the first set of boxes show how target sequences are added to the list of sequences that have a footprint determined (See “B” in Figure 17), while other sequences are passed immediately into the primer set pool (e.g. PDPass, those sequences that have been previously processed and shown to work together without forming Primer dimers or having reactivity to FRET sequences), as well as DimerTest entries (e.g. pair or primers a user wants to use, but that has not been tested yet for primer dimer or fret reactivity).
  • PDPass those sequences that have been previously processed and shown to work together without forming Primer dimers or having reactivity to FRET sequences
  • DimerTest entries e.g. pair or primers a user wants to use, but that has not been tested yet for primer dimer or fret reactivity.
  • the initial set of boxes leading up to "end of input” sort the sequences so they can be later processed properly.
  • the primer pool is basically cleared or “emptied” to start a fresh run.
  • the target sequences are then sent to "B” to be processed, and DimerTest pairs are sent to "C” to be processed.
  • Target sequences are sent to "B", where a user or software application determines the footprint region for the target sequence (e.g. where the assay probes will hybridize in order to detect the mutation (e.g. SNP) in the target sequence).
  • This region is generally shown in capital letters in figures, such as Figure 15B. It is important to design this region (which the user may further expand by defining that additional bases past the hybridization region be added) such that the primers that are designed fully encompass this region.
  • the software application INVADER CREATOR is used to design the INVADER oligonucleotide and downstream probes that will hybridize with the target region (although any type of program of system could be used to create any type of probes a user was interested in designing probes for, and thus determining the footprint region for on the target sequence).
  • the core footprint region is then defined by the location of these two assay probes on the target.
  • the system starts from the 5' edge of the footprint and travels in the 5' direction until the first base is reached, or until the first A or C (or G or T) is reached.
  • This is set as the initial starting point for defining the sequence of the forward primer (i.e. this serves as the initial N[l] site).
  • the sequence of the primer for the forward primer is the same as those bases encountered on the target region. For example, if the default size of the primer is set as 12 bases, the system starts with the bases selected as N[l] and then adds the next 11 bases found in the target sequences. This 12-mer primer is then tested for a melting temperature (e.g.
  • the system employs the formula 5'-N[x]-N[x-l]- -N[4]-N[3]-N[2]-N[l]-3 ', and x is initially 12. Then the system adjusts x to a higher number (e.g. longer sequences) until the pre-set melting temperature is found.
  • the next box in Figure 17 is used to determine if the primer that has been designed so far will cause primer-dimer and or fret reactivity (e.g. with the other sequences already in the pool). The criteria used for this determination are explained above. If the primer passes this step, the forward primer is added to the primer pool. However, if the forward primer fails this criteria, as shown in Figure 17, the starting point (N[l] is moved) one nucleotide in the 5' direction (or to the next A or C, or next G or T). The system first checks to make sure shifting over leaves enough room on the target sequence to successfully make a primer. If yes, the system loops back and check this new primer for melting temperature. However, if no sequence can be designed, then the target sequence is flagged as an enor (e.g. indicating that no forward primer can be made for this target).
  • the target sequence is flagged as an enor (e.g. indicating that no forward primer can be made for this target).
  • DimerTest pair passes the criteria, they are added to the primer set pool, and then the system goes back to "C” if there are more DimerTest pairs to be evaluated, or goes on to "D” if there are no more DimerTest pairs to be evaluated.
  • the pool of primers that has been created is evaluated.
  • the first step in this section is to examine the number of enor (failures) generated by this particular randomized run of sequences. If there were no enors, this set is the best set as maybe outputted to a user. If there are more than zero enors, the system compares this run to any other previous runs to see what run resulted in the fewest enors. If the cunent run has fewer enors, it is designated as the current best set. At this point, the system may go back to "A" to start the run over with another randomized set of the same sequences, or the pre-set maximum number of runs (e.g.
  • This best set of primers may then be used to generate as physical set of oligonucleotides such that a multiplex PCR reaction may be carried out.
  • the present invention provides methods, systems, software applications, computer systems, and a computer data storage medium that may be used to adjust primer concentrations relative to a first detection assay read (e.g. INVADER assay read) , and then with balanced primer concentrations come close to substantially equal concentrations of different amplicons.
  • a first detection assay read e.g. INVADER assay read
  • the concentrations for various primer pairs may be determined experimentally.
  • These detection assays can be on an anay of different sizes (384 well plates).
  • Having optimized primer pair concentrations in a single reaction vessel allows the user to conduct amplification for a plurality or multiplicity of amplification targets in a single reaction vessel and in a single step.
  • the yield of the single step process is then used to successfully obtain test result data for, for example, several hundred assays.
  • each well on a 384 well plate can have a different detection assay thereon.
  • the results of the single step mutliplex PCR reaction has amplified 384 different targets of genomic DNA, and provides you with 384 test results for each plate. Where each well has a plurality of assays even greater efficiencies can be obtained.
  • the present invention provides the use of the concentration of each primer set in highly multiplexed PCR as a parameter to achieve an unbiased amplification of each PCR product.
  • Any PCR includes primer annealing and primer extension steps.
  • high concentration of primers in the order of 1 uM ensures fast kinetics of primers annealing while the optimal time of the primer extension step depends on the size of the amplified product and can be much longer than the annealing step.
  • primer concentration By reducing primer concentration, the primer annealing kinetics can become a rate limiting step and PCR amplification factor should strongly depend on primer concenfration, association rate constant of the primers, and the annealing time.
  • the present invention has demonstrated a linear relationship between amplification efficiency and primer concentration and used this equation to balance primer concentrations of different amplicons, resulting in the equal amplification often different amplicons in PCR Primer Design Example 1.
  • This technique may be employed on any size set of multiplex primer pairs.
  • the PCR primers are unoptimized, and the INVADER assay is employed to detect the amplified products (See, Ohnishi et al., J. Hum. Genet. 46:471-7, 2001, herein inco ⁇ orated by reference.
  • the following experimental example describes the manual design of amplification primers for a multiplex amplification reaction, and the subsequent detection of the amplicons by the INVADER assay.
  • Each target sequence was selected from a set of pre-validated SNP -containing sequences, available in a TWT in-house oligonucleotide order entry database (see Figure 18).
  • Each target contains a single nucleotide polymo ⁇ hism (SNP) to which an INVADER assay had been previously designed.
  • the INVADER assay oligonucleotides were designed by the INVADER CREATOR software (Third Wave Technologies, Inc.
  • the footprint region in this example is defined as the INVADER "footprint", or the bases covered by the INVADER and the probe oligonucleotides, optimally positioned for the detection of the base of interest, in this case, a single nucleotide polymo ⁇ hism (See Figure 18).
  • About 200 nucleotides of each of the 10 target sequences were analyzed for the amplification primer design analysis, with the SNP base residing about in the center of the sequence. The sequences are shown in Figure 18.
  • Tm the melting temperature of the oligonucleotide is calculated using the nearest-neighbor model and published parameters for DNA duplex formation (Allawi and SantaLucia, Biochemistry, 36:10581 [1997], herein inco ⁇ orated by reference).
  • salt concentrations are often different than the solution conditions in which the nearest-neighbor parameters were obtained (1M NaCl and no divalent metals), and because the presence and concentration of the enzyme influence optimal reaction temperature, an adjustment should be made to the calculated T m to determine the optimal temperature at which to perform a reaction.
  • One way of compensating for these factors is to vary the value provided for the salt concentration within the melting temperature calculations. This adjustment is termed a 'salt conection'.
  • the term "salt conection" refers to a variation made in the value provided for a salt concentration for the purpose of reflecting the effect on a T m calculation for a nucleic acid duplex of a non-salt parameter or condition affecting said duplex.
  • N[2]-N[l] of a given oligonucleotide primer should not be complementary to N[2]-N[l] of any other oligonucleotide
  • N[3]-N[2]- N[l] should not be complementary to N[3]-N[2]-N[l] of any other oligonucleotide. If these criteria were not met at a given N[l], the next base in the 5' direction for the forward primer or the next base in the 3' direction for the reverse primer will be evaluated as an N[l] site.
  • a C rich regions were targeted in order to mirtimize the complementarity of 3' ends.
  • an INVADER assay was performed following the multiplex amplification reaction. Therefore, a section of the secondary INVADER reaction oligonucleotide (the FRET ohgonucleotide sequence) was also inco ⁇ orated as criteria for primer design; the amplification primer sequence should be less than 80%) homologous to the specified region of the FRET oligonucleotide.
  • the output primers for the 10-plex multiplex design are shown in Figure 18). All primers were synthesized according to standard oligonucleotide chemistry, desalted (by standard methods) and quantified by absorbance at A260 and diluted to 50 ⁇ M concentrated stock. Multiplex PCR was then carried out using 10-plex PCR using equimolar amounts of primer (0.01 uM/primer) under the following conditions; 100 mM KC1, 3 mM MgCl, lOmM Tris pH8.0, 200uM dNTPs, 2.5U taq, and lOng of human genomic DNA (hgDNA) template in a 50ul reaction. The reaction was incubated for (94C/30sec, 50C/44sec.) for 30 cycles.
  • the multiplex PCR reaction was diluted 1:10 with water and subjected to INVADER analysis using INVADER Assay FRET Detection Plates, 96 well genomic biplex, lOOng CLEAVASE VIII, INVADER assays were assembled as 15ul reactions as follows; lul of the 1:10 dilution of the PCR reaction, 3ul of PPI mix, 5ul of 22.5 mM MgC12, 6ul of dH20, covered with 15ul of Chillout. Samples were denatured in the INVADER biplex by incubation at 95C for 5min., followed by incubation at 63 C and fluorescence measured on a Cytofluor 4000 at various timepoints.
  • the FOZ values of the INVADER assay can be used to estimate amplicon abundance.
  • FOZm represents the sum of RED_FOZ and FAM_FOZ of an unknown concentration of target incubated in an INVADER assay for a given amount of time (m).
  • Equation la ((EOZ» - l) *500/(EOZ24o -i)) * (240/77.) ⁇ 2 (equation la)
  • equation la' is used in the calculation of the amplification factor E for the 10-plex PCR (both with equimolar amounts of primer and optimized concentrations of primer), with the value of D representing the dilution factor of the PCR reaction.
  • E> 0.3333.
  • FOZ F ((FOZm - 2) * 500 /(FOZ 24 - 2) * D) * (240 / m) A 2 (equation lb) It should be noted that in order for the estimation of amplification factor F to be more accurate, FOZ values should be within the dynamic range of the instrument on which the reading are taken. In the case of the Cytofluor 4000 used in this study, the dynamic range was between about 1.5 and about 12 FOZ.
  • primer concentration and amplification factor In order to determine the relationship between primer concentration and amplification factor (F), four distinct uniplex PCR reactions were run at using primers 1117-70- 17 and 1117- 70-18 at concentrations of O.OluM, 0.012 uM, 0.014 uM, 0.020 uM respectively.
  • the four independent PCR reactions were carried out under the following conditions; lOOmM KC1, 3mM MgCl, lOmM Tris pH 8.0, 200uM dNTPs using lOng of hgDNA as template. Incubation was carried out at (94C/30 sec, 50C/20 sec.) for 30 cycles. Following PCR, reactions were diluted 1:10 with water and run under standard conditions using INVADER Assay FRET Detection
  • Section 4.Calculation of Apparent Primer Concentrations from a Balanced Multiplex Mix As described in a previous section, primer concentration can directly influence the amplification factor of given amplicon. Under conditions of equimolar amounts of primers, FOZm readings can be used to calculate the "apparent" primer concentration of each amplicon using equation 2. Replacing Y in equation 2 with log(F) of a given amplification factor and solving for X, gives an "apparent" primer concentration based on the relative abundance of a given amplicon in a multiplex reaction. Using equation 2 to calculate the "apparent" primer concentration of all primers (provided in equimolar concenfration) in a multiplex reaction, provides a means of normalizing primer sets against each other.
  • each of the "apparent" primer concentrations should be divided into the maximum apparent primer concentration (X ma ⁇ ), such that the sfrongest amplicon is set to a value of 1 and the remaining amplicons to values equal or greater than 1
  • R[n] Xmax/X[n] (equation 3)
  • the values of R[n] are multiplied by a constant primer concentration to provide working concentrations for each primer in a given multiplex reaction, fri the example shown, the amplicon conesponding to SNP assay 41646 has an R[n] value equal to 1. All of the R[n] values were multiplied by O.OluM (the original starting primer concentration in the equimolar multiplex per reaction) such that lowest primer concentration is R[n] of 41646 which is set to 1, or O.OluM. The remainder of the primer sets were also proportionally increased as shown in Figure 21. The results of multiplex PCR with the "optimized" primer mix are described below.
  • Section 5 Using optimized primer concentrations in multiplex PCR, variation in FOZ's among 10 INVADER assays are greatly reduced.
  • sequences of less than 200 nucleotides in length were obtained with SNP annotated using brackets to indicate the SNP position for each sequence (e.g. NNNNNl ⁇ [N (Wt) /N( mt )]NNNNNNNN).
  • sequences were expanded to approximately lkB in length (500 nts flanking each side of the SNP) using BLAST analysis.
  • 16 could not expanded by BLAST, resulting in a final set of 128 sequences expanded to approximately lkB length (See, Figure 23).
  • the output file (see Figure 24, bottom of each sheet shows footprint region in upper case letters and SNP in brackets) contained 128 primer sets (256 primers, See Figure 25), four of which were thrown out due to excessively long primer sequences (SNP # 47854, 47889, 54874, 67396), leaving 124 primers sets (248 primers) available for synthesis.
  • the remaining primers were synthesized using standard procedures at the 200nmol scale and purified by desalting. After synthesis failures, 107 primer sets were available for assembly of an equimolar 107-plex primer mix (214 primers, See Figure 25). Of the 107 primer sets available for amplification, only 101 were present on the INVADER MAP plate to evaluate amplification factor.
  • Multiplex PCR was carried out using 101-plex PCR using equimolar amounts of primer (0.025uM/primer) under the following conditions; lOOmMKCl, 3mM MgCl, lOmM Tris pH8.0, 200uM dNTPs, and lOng of human genomic DNA (hgDNA) template in a 50ul reaction. After denaturation at 95C for lOmin, 2.5 units of Taq was added and the reaction incubated for (94C/30sec, 50C/44sec.) for 50 cycles. After incubation, the multiplex PCR reaction was diluted 1 :24 with water and subjected to INVADER assay analysis using INVADER MAP detection platform.
  • amplification factor F and R[n] were calculated for each of the 101 amplicons ( Figure 28).
  • R[nmax] was set at 1.6, which although Low end conections were made for amplicons which failed to provide sufficient FOZm signal at 160 min., assigning an arbitrary value of 12 for R[n].
  • High end conections for amplicons whose FOZm values at the 10 min. read, an R[n] value of 1 was arbitrarily assigned.
  • Optimized primer concentrations of the 101-plex were calculated using the basic principles outlined in the 10-plex example and equation lb, with an R[n] of 1 conesponding to 0.025uM primer (see Fig.15 for various primer concentrations).
  • Multiplex PCR was under the following conditions; lOOmMKCl, 3mM MgCl, lOmM Tris pH8.0, 200uM dNTPs, and lOng of human genomic DNA (hgDNA) template in a 50ul reaction. After denaturation at 95C for lOmin, 2.5 units of Taq was added and the reaction incubated for (94C/30sec, 50C/44sec.) for 50 cycles. After incubation, the multiplex PCR reaction was diluted 1 :24 with water and subjected to INVADER analysis using INVADER MAP detection platform.
  • Figure 32 shows one protocol for multiplex optimization.
  • genomic DNA that contains a target sequence to be analyzed by the detection assay is used as a starting material for the detection assay.
  • it may be desirable to amplify the one or more regions of the genomic DNA e.g., to generate a plurality of target sequences to be detected.
  • the present invention is not limited by the nature of the amplification technology employed. Amplification techniques include, but are not limited to, PCR and the technologies disclosed in U.S. Pat. Nos.
  • Rubicon OmniPlex technology is employed for sample preparation. Rubicon OmniPlex technology (See e.g., U.S. Pat. No. 6,197,557, herein inco ⁇ orated by reference in its entirety) reformats naturally occurring chromosomes into new molecules called Plexisomes.
  • Plexisomes represent the complete genome as amplifiable DNA units of equal length that function as a molecular relational database from which the genetic information can be more quickly and accurately recovered.
  • Use of the technology avoids PCR amplification for sample preparation and for genotyping and haplotyping for gene discovery, pharmacogenomics, and diagnostics by providing highly multiplexing and sample amplification.
  • all the various components for running any of these sample preparation methods are included in a kit (e.g. with at least a portion of a detection assay).
  • the present invention provides a high-throughput detection assay production system, allowing for high-speed, efficient production of thousands of detection assays.
  • the high- throughput production systems and methods allow sufficient production capacity to facilitate full implementation of the funnel process described above — allowing comprehensive of all known (and newly identified) markers.
  • Figure 98 shows a general overview of the oligonucleotide production and processing systems of the present invention.
  • oligonucleotides and/or other detection assay components are synthesized.
  • oligonucleotide synthesis is performed in an automated and coordinated manner.
  • produced detection assay are tested against a plurality of samples representing two or more different individuals or alleles (e.g., samples containing sequences from individuals with different ethnic backgrounds, disease states, etc.) to demonstrate the viability of the assay with different individuals.
  • the systems of the present invention allow at least 300 detection assays to be produced per day. In other embodiments, the systems of the present invention allow at least 1000, or at least 2000 detection assays to be produced per day.
  • the present invention provides an automated DNA production process.
  • the automated DNA production process includes an oligonucleotide synthesizer component and an oligonucleotide processing component.
  • the oligonucleotide production component includes multiple components, including but not limited to, an oligonucleotide cleavage and deprotection component, an oligonucleotide purification component, an oligonucleotide dry down component; an oligonucleotide de-salting component, an oligonucleotide dilute and fill component, and a quality confrol component.
  • the automated DNA production process of the present invention further includes automated design software and supporting computer terminals and connections, a product tracking system (e.g., a bar code system), and a centralized packaging component.
  • a product tracking system e.g., a bar code system
  • a centralized packaging component e.g., a product tracking system
  • the components are combined in an integrated, centrally controlled, automated production system.
  • the present invention thus provides methods of synthesizing several related oligonucleotides (e.g., components of a kit) in a coordinated manner.
  • the automated production systems of the present invention allow large-scale automated production of detection assays for numerous different target sequences.
  • detection assays are produced in an in-line fashion, such that the synthesized and processed oligonucleotides remain in the same columns and/same holder (e.g. 96 or 384 well plate). In this regard, human and machine interaction with the oligonucleotides being manufactured is minimized.
  • the various production components are grouped at a single manufacturing location.
  • the various components are not grouped.
  • the Inventory Control component may be in one location (e.g. closer to a base of customers, or closer to a particular supplier) while the synthesis components are in another location, and many of the processing components are in a third location.
  • This type of remote manufacturing is made possible, for example, by the data management systems of the present invention that allow product orders and inventory for individual assays, and individual components of assays to be tracked.
  • the production and processing facilities may be grouped for ease of use, but there may be multiple locations each producing a different component of an assay. Again, the data management systems of the present invention allow these assay components be separately tracked and assembled in finished assays.
  • sequences are sent (e.g., electronically) to a Mgh-throughput oligonucleotide synthesizer component.
  • the high-throughput synthesizer component contains multiple DNA synthesizers.
  • the synthesizers are ananged in banks.
  • a given bank of synthesizers may be used to produce one set of oligonucleotides (e.g., for an INVADER or PCR reaction).
  • the present invention is not limited to any one synthesizer.
  • synthesizers include, but not limited to MOSS EXPEDITE 16-channel DNA synthesizers (PE Biosystems, Foster City, CA), OligoPilot (Amersham Pharmacia,), the 3900 and 3948 48-Channel DNA synthesizers (PE Biosystems, Foster City, CA), POLYPLEX (Genemachines), 8909 EXPEDITE, Blue Hedgehog (Metabio), MerMade (BioAutomation, Piano, Texas), Polygen (Distribio, France), PrimerStation 960 (Intelligent Bio-Instruments, Cambridge, MA), and the Wgh-throughput synthesizer described in PCT Publication WO 01/41918.
  • synthesizers are modified or are wholly fabricated to meet physical or performance specifications particularly prefened for use in the synthesis component of the present invention.
  • two or more different DNA synthesizers are combined in one bank in order to optimize the quantities of different oligonucleotides needed. This allows for the rapid synthesis (e.g., in less than 4 hours) of an entire set of oligonucleotides (all the oligonucleotide components needed for a particular assay, e.g., for detection of one SNP using an INVADER assay).
  • the synthesizers are configured for generating oligonucleotides in 96 or 384 well plates.
  • the DNA synthesizer component includes at least 100 synthesizers. In other embodiments, the DNA synthesizer component includes at least 200 synthesizers. In still other embodiments, the DNA synthesizer component includes at least 250 synthesizers. In some embodiments, the DNA synthesizers are run 24 hours a day.
  • the present invention provides nucleic acid synthesizers and methods of using and modifying nucleic acid synthesizers.
  • the present invention provides highly . efficient, reliable, and safe synthesizers that find use, for example, in high throughput and automated nucleic acid synthesis (e.g. anays of synthesizers), as well as methods of modifying pre-existing synthesizers to improve efficiency, reliability, and safety.
  • a problem with cunently available synthesizers is the emission of undesirable gaseous or liquid materials that pose health, environmental, and explosive hazards. Such emissions result from both the normal operation of the instrument and from instrument failures. Emissions that result from instrument failures cause a reduction or loss of synthesis efficiency and can provoke further failures and/or complete synthesizer failure. Conection of failures may require taking the synthesizer off-line for cleaning and repair.
  • the present invention provides nucleic acid synthesizers with components that reduce or eliminate unwanted emissions and that compensate for and facilitate the removal of unwanted emissions, to the extent that they occur at all.
  • the present invention also provides waste handling systems to eliminate or reduce exposure of emissions to the users or the environment. Such systems find use with individual synthesizers, as well as in large-scale synthesis facilities comprising many synthesizers (e.g. anays of synthesizers).
  • the present invention provides efficient and safe "open system synthesizers.”
  • Open system synthesizers are contrasted to "closed system synthesizers" in that the reagent delivery, synthesis compartments, and waste exfraction for each synthesis column are not contained in a system that remains physically closed (i.e., closed from both the ambient environment and from the other synthesis columns in the same instrument) for the duration of the synthesis run.
  • tubing or other means
  • the dispensing and/or removal of reagent may be through means that are not physically coupled to the reaction compartment.
  • a common dispensing or waste removal means may be shared by multiple reaction compartments, such that each compartment sharing the means is serviced in turn.
  • An example of an "open system synthesizer" is described in PCT Publication WO 99/65602, herein inco ⁇ orated by reference in its entirety. This publication describes a rotary synthesizer for parallel synthesis of multiple oligonucleotides. The tubing that supplies the synthesis reagents to the synthesis column does not form a continuous closed seal to the synthesis columns. Instead, the rotor turns, exposing the synthesis columns, in series, to the dispense lines, which inject synthesis reagents into the synthesis column.
  • Open synthesizers offer advantages over closed synthesizers for the simultaneous production of multiple oligonucleotides. For example, a large number of independent synthesis columns, each intended to produce a distinct ohgonucleotide, are exposed to a smaller number of dedicated reagent dispensers (e.g., four dedicated dispensers for each of the nucleotides). Open systems also provide easy access to synthesis columns, which can be added or removed without detaching any otherwise fixed connections to reagent dispensing tubing.
  • oligonucleotide synthesis involves the use of an anay of hazardous materials, including but not limited to methylene chloride, pyridine, acetic anhydride, 2,6-lutidine, acetonitrile, tefrahydrofurane, and toluene.
  • hazardous materials including but not limited to methylene chloride, pyridine, acetic anhydride, 2,6-lutidine, acetonitrile, tefrahydrofurane, and toluene.
  • hazardous materials including but not limited to methylene chloride, pyridine, acetic anhydride, 2,6-lutidine, acetonitrile, tefrahydrofurane, and toluene.
  • These reagents can have a variety of harmful effects on those who may be exposed to them. They can be mildly or extremely irritating or toxic upon short-term exposure; several are more severely toxic and or carcinogenic with long-term exposure. Many can create
  • Emission or leakage of reagents during operation can have consequences beyond risks to personnel and to the environment.
  • instruments may need to be removed from operation for cleaning, leading to a temporary decrease in production capacity of a synthesis facility.
  • any emission or leakage may cause damage to parts of the instrument or to other instruments or aspects of the facility, necessitating repair or replacement of any such parts or aspects, increasing the time and cost of bringing an instrument back into operation.
  • the synthesizers of the present invention provide a number of novel features that dramatically improve synthesizer performance and safety compared to available synthesizers. These novel features work both independently and in conjunction to provide enhanced performance.
  • the synthesizers of the present invention prevent loss of pressure during synthesis and waste disposal. By preventing loss of pressure, synthesis columns are purged properly and do not overflow during subsequent synthesis steps. Thus, prevention of pressure loss further prevents liquid overflow and instrument contamination. Additionally, in some embodiments, sufficient pressure differentials are maintained across all columns to allow efficient synthesis and purging without instrument failure.
  • the present invention provides methods for modifying existing synthesizers to improve their efficiency. For example, one or more of the novel components of the present invention may be added into or substituted into existing synthesizers to improve efficiency and performance.
  • the present invention further provides means of reducing exposure of operators and the environment to synthesis reagents and waste. In one embodiment, the present invention reduces exposure by improving collection and disposal of emissions that occur during the normal operation of various synthesis instruments. In another embodiment, the present invention reduces exposure by improving aspects of the instrument to reduce risk of malfunctions leading to reagent escape from the system, e.g., through leakage, overflow or other spillage.
  • the present invention provides open-system solid phase synthesizers that are suitable for use in large-scale polymer production facilities. Each synthesizer is itself capable of producing large volumes of polymers. However, the present invention provides systems for integrating multiple synthesizers into a production facility, to further increase production capabilities.
  • Figure 33 illustrates a synthesizer 1.
  • the synthesizer 1 is designed for building a polymer chain by sequentially adding polymer units to a solid support in a liquid reagent.
  • the liquid reagents used for synthesizing oligonucleotides may vary, as the successful operation of the present invention is not limited to any particular coupling chemistry.
  • liquid reagents include, but are not limited to: Acetonitrile (wash); 2.5% dichloroacetic acid in methylene chloride (deblock); 3% tetrazole in acetonitrile (activator); 2.5% cyanoethyl phosphoramidite in acetonitrile (A, C, G, T); 2.5% iodine in 9% water, 0.5% pyridine, 90.5% THF (oxidizer); 10% acetic anhydride in tefrahydrofuran (CAP A); and 10% 1-methylimidazole, 10% pyridine, 80% THF.
  • Various useful reagents and coupling chemistries are described in U.S. Pat. 5,472,672 to Bennan, and U.S. Pat. No. 5,368,823 to McGraw et al. (both of which are herein inco ⁇ orated by reference in their entireties).
  • the solid support generally resides within a synthesis column and various hquid reagents are sequentially added to the synthesis column. Before an additional liquid reagent is added to a synthesis column, the previous liquid reagent is preferably purged from the synthesis column.
  • the synthesizer 1 is particularly suited for building nucleic acid sequences, the synthesizer 1 is also configured to build any other desired polymer chain or organic compound (e.g. peptide sequences).
  • the synthesizer 1 preferably comprises at least one bank of valves and at least one bank of synthesis columns. Within each bank of synthesis columns, there is at least one synthesis column for holding the solid support and for containing a liquid reagent such that a polymer chain can be synthesized. Within the bank of valves, there are preferably a plurality of valves configured for selectively dispensing a liquid reagent into one of the synthesis columns. The synthesizer 1 is preferably configured to allow each bank of synthesis columns to be selectively purged of the presently held liquid reagent.
  • the synthesizer of the present invention is configured to allow synthesis columns within a bank to be purged even when not all of the synthesis columns contain liquid reagents (e.g. only a portion of the synthesis columns in a bank received a liquid reagent (i.e. "active"), while the remaining synthesis columns are no longer receiving liquid reagent (i.e. "idle").
  • the design of the material in the synthesis columns allows idle columns to resist the downward pressure of gas, thus making this pressure available to purge the synthesis columns that contain liquid reagent.
  • Additional banks of valves provide the synthesizer 1 with greater flexibility.
  • each bank of valves can be configured to distribute liquid reagents to a particular bank of synthesis columns in a parallel fashion to minimize the processing time.
  • Multiple banks of valves can also be configured to distribute liquid reagents to a particular bank of synthesis columns in series. This allows the synthesizer 1 to hold a larger number of different reagents, thus being able to create varied nucleic acid sequences (e.g. 48 oligonucleotides, each with a unique sequence).
  • Figure 33 illustrates a top view of a rotary synthesizer 1.
  • the synthesizer 1 includes a base 2, a cartridge 3, a first bank of synthesis columns 4, a second bank of synthesis columns 5, a plurality of dispense lines 6, a plurality of fittings 7 (a first bank of fittings 13, and a second bank of fittings 14), a first bank of valves 8 and a second bank of valves 9.
  • Each of the valves is capable of selectively dispensing a liquid reagent into one of the synthesis columns.
  • Each of the synthesis columns is preferably configured for retaining a solid support such as polystyrene or CPG and holding a liquid reagent. Further, as each liquid reagent is sequentially deposited within the synthesis column and sequentially purged therefrom, a polymer chain is generated (e.g. nucleic acid sequence).
  • each of the valves within the first bank and second bank of valves 8 and 9, is coupled to a conesponding reservoir.
  • Each of the plurality of reservoirs is pressurized (e.g. by argon gas).
  • argon gas a particular liquid reagent from the conesponding reservoir is dispensed to a conesponding synthesis column.
  • Each of the plurality of dispense lines 6 is coupled to a conesponding one of the valves within the first and second banks of valves 8 and 9.
  • Each of the plurality of dispense lines 6 provides a conduit for fransferring a liquid reagent from the valve to a conesponding synthesis column.
  • Each one of the plurality of dispense lines 6 is preferably configured to be flexible and semi-resilient in nature.
  • the dispense lines of the present invention have a large bore size to prevent clogging.
  • the internal diameter of the dispense tube is at least 0.25mm. In other embodiments, the internal diameter of the tube- is at least 0.50mm or at least 0.75mm. fri some embodiments, the internal diameter of the tube is greater than or equal to 1.0mm (e.g. 1.0mm, or 1.2mm, or 1.4mm).
  • the plurality of dispense lines 6 are each made of a material such as PEEK, glass, or coated with TEFLON or Parlene, or coated/uncoated stainless steel or other metallic material.
  • a material such as PEEK, glass, or coated with TEFLON or Parlene, or coated/uncoated stainless steel or other metallic material.
  • useful characteristics of the material used for the dispense lines would be resistance to degradation by the liquid reagents, minimal "wetting" by the liquid reagents, ease of fabrication, relative rigidity, and ability to be produced with a smooth surface finish.
  • Metallic tubing e.g. stainless steel
  • benefit from elecfropolishing to improve the surface finish e.g. in coated or uncoated application.
  • Another important characteristic of useful dispense lines in the ability to provide a seal between the plurality of valves 10 and the plurality of fittings 7.
  • Each of the plurality of fittings 7 is preferably coupled to one of the plurality of dispense lines 6.
  • the plurality of fittings 7 are preferably configured to prevent the reagent from splashing outside the synthesis column as the reagent is dispensed from the fitting to a particular synthesis column positioned below the fitting.
  • the fitting includes a nozzle that prevents reagents from drying at the point fluid exits the nozzle (e.g. prevents dried reagents from causing the reagents stream to dispense at angles away from the intended synthesis column). Construction techniques to achieve consistent flow at the discharge point of the liquid reagents is achieved by the use of high quality parts and construction.
  • a drawn tip For example, clean square cuts (without buns or shavings), or the use of a "drawn tip" (i.e., a tip of reduced diameter at the discharge point).
  • the use of a drawn tip reduces the wall thickness at the point of discharge, thus reducing the area of the tube wall cross section, providing a smooth transition from the larger portion of the tube (reducing flow resistance) and increases the likelihood of a clean separation of the discharged liquid reagent from the tip of the tube.
  • This clean "snap" of the liquid reagent minimizes the retention of the discharged fluid at the tip, and thus minimizes subsequent build up of any solids (e.g. dried reagent).
  • the fluid front will actually reside within the confines of the tube after discharge of the desired volume. This minimizes surface evaporation and helps to maintain a clean orifice (e.g. prevent reagent from drying at the tip).
  • Another example of a useful technique to prevent liquid reagent from drying at the discharge point is providing a sleeve or sheath over the dispense line to a point near the tip (dispense point). This sleeve or sheath is particularly useful when employed in conjunction with a relatively flexible dispense line.
  • the first and second banks of valves 8 and 9 each have thirteen valves.
  • the number of valves in each bank is merely for exemplary pmposes (e.g. other numbers ofvalves may be employed, like 14, 15, 16, 17, etc.).
  • Each of the synthesis columns within the first bank of synthesis columns 4 and the second bank of synthesis columns 5 is presently shown resting in one of a plurality of receiving holes 11 within the cartridge 3.
  • each of the synthesis columns within the conesponding plurality of receiving holes 11 is positioned in a substantially vertical orientation.
  • Each of the synthesis columns is configured to retain a solid support such as polystyrene or CPG and hold liquid reagent(s). In prefened embodiments, polystyrene is employed as the solid support. Alternatively, any other appropriate solid support can be used to support the polymer chain being synthesized.
  • each of the valves selectively dispenses a liquid reagent through one of the plurality of dispense lines 6 and fittings 7.
  • the first and second banks of valves 8 and 9 are preferably coupled to the base 2 of the synthesizer 1.
  • the cartridge 3 which contains the plurality of synthesis columns 12 rotates relative to the synthesizer 1 and relative to the first and second banks of valves 8 and 9. By rotating the cartridge 3, a particular synthesis column 12 is positioned under a specific valve such that the conesponding reagent from this specific valve is dispensed into this synthesis column.
  • the cartridge 3 has a home position that allows the synthesizer to be properly aligned before operation (such that the liquid reagent is properly dispensed into the synthesis columns).
  • the first and second banks of valves 8 and 9 are capable of simultaneously and independently dispensing liquid reagents into conesponding synthesis columns.
  • FIG. 34 A cross sectional view of synthesizer 1 is depicted in Figure 34.
  • the synthesizer 1 includes the base 2, a set of valves 15, a motor 16, a gearbox 17, a chamber bowl 18, a drain plate 19, a drain 20, a cartridge 3, abottom chamber seal 21, amotor connector 22, a waste tube system 23, a controller 24, and a clear window 25.
  • the valves 15 are coupled to base 2 of the synthesizer 1 and are preferably positioned above the cartridge 3 around the outside edge of the base 2.
  • This set of valves 15 preferably contains fifteen individual valves which each deliver a conesponding liquid reagent in a specified quantity to a synthesis column held in the cartridge 3 positioned below the valves.
  • Each of the valves may dispense the same or different liquid reagents depending on the user-selected configuration.
  • the set of valves 15 is capable of simultaneously dispensing a reagent to multiple synthesis columns within the cartridge 3.
  • each one of the valves 15 is capable of dispensing a conesponding liquid reagents to any one of the synthesis columns within the cartridge 3.
  • the synthesizer 1 may have multiple sets of valves.
  • the plurality of valves within the multiple sets of valves may be configured in a variety of ways to dispense the liquid reagents to a select one or more of the synthesis columns.
  • the synthesizer 1 is capable of simultaneously dispensing the same reagent in parallel from multiple sets of valves to conesponding banks of synthesis columns.
  • the multiple banks of synthesis columns may be processed in parallel.
  • each individual valve within multiple sets of valves may contain entirely different liquid reagents such that there is no duplication of reagents among any individual valves in the multiple sets of valves. This configuration allows the synthesizer 1 to build polymer chains requiring a large variety of reagents without changing the reagents associated with each valve.
  • the motor 16 is preferably mounted to the base 2 through the gear box 17 and the motor connector 22.
  • the chamber bowl 18 preferably sunounds the motor connector 22 and remains stationary relative to the base 2.
  • the chamber bowl 18 is designed to hold any reagent spilled from the plurality of synthesis columns 12 during the purging process (or the dispensing process). Further, the chamber bowl 18 is configured with a tall shoulder to insure that spills are contained within the bowl 18.
  • the bottom chamber seal 21 preferably provides a seal around the motor connector 22 in order to prevent the contents of the chamber bowl 18 from flowing into the gear box 17 (see Figure 34).
  • the bottom chamber seal 21 is preferably composed of a flexible and resilient material such as TEFLON (or elastomer which conforms to any inegularities of the motor connector 22). Alternatively, the bottom chamber seal can be composed of any other appropriate material. In particularly prefened embodiments, the bottom chamber seal is composed of material that resists constant contact with liquid reagents (e.g., TEFLON or Parlene).
  • the bottom chamber seal 21 may have frictionless properties that allow the motor connector 22 to rotate freely within the seal. For example, coating this flexible material with TEFLON helps to achieve a low coefficient of friction.
  • the clear window 25 is attached to (formed in) a top cover 30 of the synthesizer 1 and covers the area above the cartridge 3.
  • the top cover 30 of synthesizer 1 seals the top part of the chamber (when in place), and opens up allowing an operator or maintenance person access to the interior of the synthesizer 1.
  • the clear window 25 in top cover 30 allows the operator to observe the synthesizer 1 in operation while providing a pressure sealed environment within the interior of the synthesizer 1.
  • the clear window 25 also includes a gas fitting 27 attached therethrough.
  • the gas fitting 27 is coupled to a gas line 28.
  • the gas line 28 preferably continuously emits a stream of inert gas (e.g. Argon) which flows into the synthesizer 1 through the gas fitting 27 and flushes out traces of air and water from the plurality of synthesis columns 12 within the synthesizer 1.
  • Providing the inert gas flow through the gas fitting 27 into the synthesizer 1 prevents the polymer chains being formed within the synthesis columns from being contaminated without requiring the plurality of synthesis columns 12 to be hermetically sealed and isolated from the outside environment.
  • Figure 35 shows the cartridge 3 in chamber bowl 18, with the top plate 30 removed, thus revealing the top chamber seal 31.
  • Top chamber seal 31 is designed to provide a tight seal between top plate 30 and chamber bowl 18, such that inert gas applied through clear window 25 does not leak. If the top chamber seal 31 does not function properly, the inert gas leaks out (lowering the pressure in the chamber), thus causing the purge operation (that relies on the pressure on the inert gas) to fail. When the purge operation fails, un-purged columns quickly fill up and overflow.
  • a V-seal type top chamber seal is employed to prevent leakage of gas.
  • the hinges and latches on top plate 30 are precisely machined to provide balanced forces on the top plate 30, such that the top plate 30 fits tightly over the chamber bowl.
  • Figure 36 illusfrates a detailed view of a cartridge 3 for synthesizer 1.
  • the cartridge 3 is circular in shape such that it is capable of rotating in a circular path relative to the base 2 and the first and second banks of valves 8 and 9.
  • the cartridge 3 has a plurality of receiving holes 11 on its upper surface around the peripheral edge of the cartridge 3. Each of the plurality of receiving holes 11 is configured to hold one of the synthesis columns 12.
  • the plurality of receiving holes 11, as shown on the cartridge 3, is divided up among four banks.
  • a bank 32 illustrates one of the four banks on the cartridge 3 and contains twelve receiving holes, wherein each receiving hole is configured to hold a synthesis column.
  • An exemplary synthesis column 12 is shown being inserted into one of the plurality of receiving holes 11.
  • the total number of receiving holes shown on the cartridge 3 includes forty-eight (48) receiving holes, divided into four banks of twelve receiving holes each.
  • the number of receiving holes and the configuration of the banks of receiving holes is shown on the cartridge 3 for exemplary pu ⁇ oses only. Any appropriate number of receiving holes and banks of receiving holes can be included in the cartridge 3.
  • the receiving holes 11 within the cartridge each have a precise diameter for accepting the synthesis columns 12, which also each have a conesponding precise exterior surface 61 (see Figure 44) to provide a pressure-tight seal when the synthesis columns 12 are inserted into the receiving holes 11.
  • the synthesis column includes a column seal 65 (see Figure 44), such as a ring seal or a ball seal (e.g., a flexible TEFLON ring that flexes on engagement of the synthesis column in the receiving hole 11).
  • a seal such as a ring seal, is provided above or in the receiving holes 11 (see, e.g., Figure 44).
  • Figure 37 depicts an exemplary drain plate 19 of the synthesizer 1.
  • the drain plate 19 is coupled to the motor connector 22 (not shown) through securing holes 33. More specifically, the drain plate 19 is attached to the motor connector 22, which rotates the drain plate 19 while the motor 16 is operating and the gear box 17 is turning.
  • the cartridge 3 and the drain plate 19 are preferably configured to rotate as a single unit.
  • the drain plate 19 is configured to catch and direct the liquid reagents as the liquid reagents are expelled from the plurality of synthesis columns (during the purging process).
  • the motor 16 is configured to rotate both the cartridge 3 and the drain plate 19 through the gear box 17 and the motor connector 22.
  • the bottom chamber seal 21 allows the motor connector 22 to rotate the cartridge 3 and the drain plate 19 through a portion of the chamber bowl 18 while still containing spilled reagents in the chamber bowl 18.
  • the controller 24 is coupled to the motor 16 to activate and deactivate the motor 16 in order to rotate the cartridge 3 and the drain plate 19.
  • the controller 24 (see Figure 34) provides embedded control to the synthesizer and controls not only the operation of the motor 16, but also the operation of the valves 15 and the waste tube system 23.
  • the drain plate 19 has a plurality of securing holes 33 for attaching to the motor connector 22.
  • the drain plate 19 also has a top surface 34 which may, in some embodiments, attach to the underside of the cartridge 3.
  • a drain plate gasket is provided between the drain plate 19 and cartridge 3 (see below).
  • the cartridge 3 holds the plurality of synthesis columns grouped into a plurality of banks.
  • the drain plate preferably has a collection area conesponding to each of the banks of synthesis columns (e.g. four in Figure 37 to conespond to the four banks of synthesis columns in cartridge 3).
  • Each of these four collection areas 35, 36, 37 and 38 in Figure 37 forms a recessed area below the top surface 34 and is designed to contain and direct material flushed from the synthesis columns within the bank above the collection area.
  • Each of the four collection areas 35, 36, 37 and 38 is positioned below a conesponding one of the banks of synthesis columns on the cartridge 3.
  • the drain plate 19 is rotated with the cartridge 3 to keep the conesponding collection area below the conesponding bank.
  • FIG 37 there are four drains 39, 40, 41, and 42 each of which is located within one of the four collection areas 35, 36, 37 and 38 respectively.
  • the collection areas are configured to contain material flushed from conesponding synthesis columns and pass that material through the drains.
  • any appropriate number of collection areas and drains can be included within a drain plate.
  • Figure 38 A shows a top view of drain plate gaskets 43.
  • the drain plate gasket is configured to be situated between drain plate 19 and cartridge 3. Drain plate gasket 43 is shown in Figure 38 A with guide holes 44 and drain cutouts 57, 58, 59, and 60.
  • Drain cut-outs 57-60 allow the bottom column opening of synthesis columns 12 to discharge material into collection areas 35-38 in drain plate 19.
  • the drain cut outs minor the receiving holes in the cartridge (see cut-outs 60 in Figure 38B), such that each column is able to discharge material into collection areas 35-38, while having a seal around each synthesis column.
  • all of the cut-outs are for the synthesis columns, like the cuts 60 depicted in Figure 38B.
  • the drain plate gaskets of the present invention may be made of any suitable material (e.g. that will provide a tight seal above drain plate 19, such that gas and liquid do not escape).
  • the drain plate gasket is composed of rubber. Providing a tight seal between cartridge 3 and drain plate 19 with a drain plate gasket helps maintain the proper pressure of inert gas during purging procedures, such that synthesis columns with liquid reagent properly drain (preventing overflow during the next cycle).
  • the seal between cartridge 3 and drain plate 19 may also be improved by the addition of grease between the components, or very finely machining the contact points between the two components.
  • the seal between the cartridge and drain plate is improved by physically bonding the plates together, or machining either the cartridge or drain plate such that concentric ring seals may inserted into the machined component.
  • the two components are manufactured as a single component (e.g. a single components with all the features of both the cartridge and drain plate formed therein).
  • one component is provided with plurality of concentric circular rings that contact the flat surface of the other component and act as seals.
  • Figure 39 shows a side view of a drain plate gasket 43 situated between cartridge 3 and drain plate 19.
  • Figure 39 also shows a drain 20 extending from drain plate 19.
  • Figure 39 also shows a drain with sealing ring 45 (sealing ring is labeled 46).
  • the sealing ring 46 tightly seals the connection between the drain 45 and the waste tube system 23 (see Figure 40).
  • a synthesis column 12 inserted in cartridge 3, passing through drain plate gasket 43, and ending in drain plate 19.
  • the waste tube system 23 is preferably utilized to provide a pressurized environment for flushing material including reagents from the plurality of synthesis columns located within a conesponding bank of synthesis columns and expelling this material from the synthesizer 1.
  • the waste tube system 23 can be used to provide a vacuum for drawing material from the plurality of synthesis columns located within a conesponding bank of synthesis columns.
  • a cross-sectional view of the waste tube system 23 is illustrated in Figure 39.
  • the waste tube system 23 comprises a stationary tube 47 and a mobile waste tube 48.
  • the stationary tube 47 and the mobile waste tube 48 are slidably coupled together.
  • the stationary tube 47 is attached to the chamber bowl 18 and does not move relative to the chamber bowl (see Figure 41).
  • the mobile tube 48 is capable of sliding relative to the stationary tube 47 and the chamber bowl 18.
  • the waste tube system 47 does not expel any reagents.
  • both the stationary tube 47 and the mobile tube 48 are preferably mounted flush with the bottom portion of the chamber bowl 18 (see Figure 41).
  • the waste tube system 23 purges the material from the conesponding bank of synthesis columns.
  • the mobile tube 48 rises above the bottom portion of the chamber bowl 18 towards the drain plate 19.
  • the drain plate 19 is rotated over to position a drain conesponding to the bank to be flushed, above the waste tube system 23.
  • the mobile tube 48 then couples to the drain (e.g., 20 or 45) and the material is flushed out of the conesponding bank of synthesis columns and into the drain plate 19.
  • the liquid reagent is purged from the conesponding bank of synthesis columns due to a sufficient pressure differential between a top opening 49 ( Figure 44) and a bottom opening 50 ( Figure 44) of each synthesis column.
  • This sufficient pressure differential is preferably created by coupling the mobile waste tube 48 to the conesponding drain.
  • the waste tube system 23 may also include a vacuum device 29 (see, Figure 34) coupled to the stationary tube 47 (see Figure 40) wherein the vacuum device 29 is configured to provide this sufficient pressure differential to expel material from the conesponding bank of synthesis columns. When this sufficient pressure differential is generated, the excess material within the synthesis columns being flushed, then flows through the conesponding drain and is carried away via the waste tube system 23.
  • the mobile tube 48 slides over the conesponding drain such that the mobile tube 48 and the drain act as a single unit.
  • the waste tube system 23 includes a mobile tube 48 which engages the conesponding drain by positioning itself directly below the drain and then sealing against the drain without sliding over the drain.
  • the mobile tube 48 may include a drain seal positioned on top of the mobile tube.
  • the mobile tube 48 is not locked to the conesponding drain. In the event that this drain is accidentally rotated while the mobile waste tube 48 is engaged with the drain, the drain and mobile tube 48 of the synthesizer 1 will simply disengage and will not be damaged.
  • the bottom of the chamber bowl 18 has a chamber drain 64 (see Figure 41) to collect and remove any spilled material in the chamber bowl.
  • material may be removed before it builds up and leaks into other parts of the synthesizer (e.g. motor 16 or gear box 17).
  • the chamber drain is in a closed position during synthesis and purging. When the top cover of the synthesizer is opened, the chamber drain can be opened, drawing out unwanted gaseous or liquid emissions (e.g., using a vacuum source). Coordination of the chamber drain opening to the top cover opening may be accomplished by mechanical or electric means.
  • the present invention selectively purges individual banks of synthesis columns such that only the synthesis columns within a selected bank or banks are purged.
  • the waste system is fitted for qualitative monitoring of detritylation. For example, colorimetric analysis of waste effluent using, for example, a CCD camera or a similar device provides a yes/no answer on a particular detritylation level. Qualitative analysis can also be accomplished by spectrophotometricly, or by testing effluent conductivity.
  • the effluent from each column is monitored when a bank of columns is purged.
  • the synthesizer 1 includes two waste tube systems 23 for flushing two banks of synthesis columns simultaneously.
  • any appropriate number of waste tube systems can be included within the synthesizer 1 for selectively flushing synthesis columns or banks of synthesis columns.
  • the waste tube systems 23 are spaced on opposite sides of the chamber bowl 18 (i.e. they are directly across from each other, see Figure 41).
  • the force on the drain plate 19 is equalized during flushing procedures (e.g. the drain plate is less likely to tip one way or the other from force being applied to just one side of the plate).
  • a single waste tube system 23 may be provided for flushing the plurality of banks of synthesis columns.
  • a balancing force be provided on the opposite side of the drain plate 19, e.g., such as would be provided by the presence of a second waste tube system 23.
  • a balancing force is provided by a dummy waste tube system (not shown), that may be actuated in the same fashion as the waste tube system 23, but which does not serve to drain the bank of synthesis columns to which it is deployed.
  • the controller 24 controls the motor 16 such that the cartridge is rotated to ahgn the conect synthesis columns with the dispense lines 6 conesponding to the appropriate valves 15 during dispensing operations and that the conect one of the drains 39, 40, 41, and 42 are aligned with an appropriate waste tube system 23 during a flushing operation.
  • the synthesizer comprises a means of delivering energy to the synthesis columns to, for example, increase nucleic acid coupling reaction speed and efficiency, allowing increased production capacity.
  • the delivery of energy comprises delivering heat to the chamber or the columns.
  • Heat may be provided by a number of means, including, but not limited to, resistance heaters, visible or infrared light, microwaves, Peltier devices, transfer from fluids or gasses (e.g., via channels or a jacketed system).
  • heat generated by another component of a synthesis or production facility system e.g., during a waste neutralization step is used to provide heat to the chamber or the columns.
  • heat is delivered through the use of one or more heated reagents. Delivery of heat also comprises embodiments wherein heat is created within the, e.g. , by magnetic induction or microwave treatment. In some embodiments, heat is created at or within synthesis columns. It is contemplated that heating may be accomplished through a combination of two or more different means.
  • the delivery of heat provides substantially uniform heating to two or more synthesis columns.
  • heating is carried out at a temperature in a range of about 20 °C to about 60 °C.
  • the present invention also provides methods for determining an optimum temperature for a particular coupling chemistry. For example, multiple synthesizers are run side-by-side with each machine run at a different temperature. Coupling efficiencies are measured and the optimum temperature for one or more incubations times are determined. In other embodiments, different amounts of heat are delivered to different synthesis columns within a single synthesizer, such that different reaction chemistries or protocols can be run at the same time.
  • the sealed system of the present invention will be configured to tolerate variations in the system pressure (i.e., the pressure within the sealed system) related to heating or other energy input to the system.
  • the system e.g., every component of the system and every junction or seal within the system
  • the system will be configured to withstand a range of pressures, e.g., pressures ranging from 0 to at least 1 arm, or about 15 psi. It is contemplated that pressures may be varied between different points within the system.
  • reagents and waste fluids are moved through the synthesis column by use of a pressure differential between one end (e.g., an input aperture) and the other (e.g., a drain aperture) of the synthesis column.
  • the system of the present invention is configured to use pressure differentials within a pressurized system (e.g., wherein a system segment having lower pressure than another system segment nonetheless has higher pressure than the environment outside the sealed system).
  • the prevention of backward flow of reagents through the system is controlled by use of pressure.
  • valves are provided to assist in confrol of the direction of flow.
  • the synthesizer comprises a mixing component configured to mix reaction components, e.g., to facilitate the penetration of reagents into the pores of the solid support.
  • Mixing may be accomplished in a number of ways. In some embodiments, mixing is accomplished by forced movement of the fluid through the matrix (e.g., moving it back and forth or circulating it through the matrix using pressure and/or vacuum, or with a fluid oscillator). Mixing may also be accomplished by agitating the contents of the synthesis column (e.g., stirring, shaking, continuous or pulsed ultra or subsonic waves). Examples are provided in Figures 42A-C, which illustrate different embodiments of energy input components 95 and mixing components 96. Also, Figures 43A-B illustrate different combinations of energy input components 95 and mixing components 96.
  • an agitator is used that avoids the creation of standing waves in the reaction mixture.
  • the agitator is configured to utilize a reaction vessel surface or reaction support surface (e.g. , a surface of a synthesis column) to serve as resonant members to transfer energy into fluid within a reaction mixture.
  • a horn is applied directly to the cartridge 3 to provided pulsed or continuous ultra sonic energy to the synthesis columns therein.
  • the matrix is an active component of the mixing system.
  • the matrix comprises paramagnetic particles that may be moved through the use of magnets to facilitate mixing.
  • the matrix is an active component of both mixing and heating systems (e.g. , paramagnetic particles may be agitated by magnetic control and heated by magnetic induction). It is contemplated that any of these mixing means may be used as the sole means of mixing, or that these mixing components may be used in combination, either simultaneously or in sequence. In prefened embodiments, the heating component and the mixing component are under automated control.
  • Figure 42 illustrates a cross sectional view of a synthesis column 12.
  • the synthesis column is an integral portion of the synthesizer 1.
  • the polymer chain is formed within the synthesis column 12. More specifically, the synthesis column 12 holds a solid support 54 on which the polymer chain is grown.
  • suitable solid supports include, but are not limited to, polystyrene, controlled pore glass, and silica glass.
  • the solid support 54 is sequentially submerged in various reagents for a predetermined amount of time. With each deposit of a reagent, an additional unit is added, or the solid support is washed, or failure sequences are capped, etc.
  • the solid support 54 is held within the synthesis column 12 by a bottom frit 55.
  • a top frit 53 is included above the solid support (e.g. to help resist downward gas pressure when the particular synthesis column does not have liquid reagents, but other synthesis columns within the bank are being purged of their liquid contents).
  • the synthesis column 12 includes a top opening 49 and a bottom opening 50. During the dispensing process, the synthesis column 12 is filled with a reagent through the top opening 49. During the purging process, the synthesis column 12 is drained of the reagent through the bottom opening 50. The bottom fiit 55 prevents the solid support from being flushed away during the purging process.
  • each synthesis column 12 fits within the receiving hole 11 within the cartridge 3 and provides a pressure tight seal around each synthesis column within the cartridge 3.
  • each synthesis column is formed of polyethylene or other suitable material.
  • the receiving holes 11 of the cartridge 3 are provided with seals, such as O-ring seals 67, that will flex on engagement of the synthesis column 12 in receiving hole 11 and accommodate any inegularities in the exterior surface 61 of the synthesis column 12, thus assuring the presence of a pressure-tight seal.
  • the material inside the synthesis column (e.g. in Figure 44, this includes top frit 53, solid support 54, and bottom frit 55) is configured to resist the downward pressure of gas (e.g. , to provide back pressure) applied during the purging process when the particular synthesis column does not have liquid reagent.
  • gas e.g. , to provide back pressure
  • other synthesis columns that do contain liquid reagents may be successfully purged with the application of gas pressure during the purging process (i.e. the synthesis columns without liquid reagent do not allow a substantial portion the gas pressure applied during the purging process to escape through their bottom openings).
  • Other packing materials may also be added to the synthesis columns to help maintain the pressure differential across the column when it is idle.
  • One method for constructing a synthesis column that successfully resists the downward pressure of gas (when no liquid reagent has been added to this column) is to include a top frit in addition to a bottom frit. Determining what type of top frit is suitable for any given synthesis column and type of solid support may be determined by test runs in the synthesizer. For example, the columns may be loaded into the synthesizer with the candidate top frit (and solid support and bottom frit), and instructions for synthesizing different length oligonucleotides inputted (i.e., this will allow certain columns to sit idle while other columns are still having liquid dispensed into them and purged out).
  • Observation through the glass panel examining the amount of leakage from overflowing columns, and testing the quality of the resulting oligonucleotides, are all methods to determine if the top frit is suitable (e.g., a thicker or smaller pore top frit may be employed if problems associated with insufficient back pressure are seen).
  • a thicker or smaller pore top frit may be employed if problems associated with insufficient back pressure are seen.
  • Another method for constructing a synthesis column that successfully resists the downward pressure of gas (when no liquid reagent has been added to this column) is to provide a ⁇ solid support that resists this downward force even when no liquid reagent is in the columns.
  • One suitable solid support material is polystyrene (e.g. US Pat. No. 5,935,527 to Andrus et al, hereby inco ⁇ orated by reference).
  • the styrene (of the polystyrene) is cross-linked with a cross-linking material (e.g. divinylbenzene).
  • the cross-linking ratio is 10-60 percent. In prefened embodiments, the cross-linking ration is 20-50 percent.
  • the cross-linking ratio is about 30-50 percent.
  • the polystyrene solid support is used in conjunction with a top frit in order to successfully resist the downward pressure of gas during the purging process.
  • the polystyrene is used as the solid support for synthesis.
  • a different support such as controlled pore glass, is used as the support for the synthesis reaction, and the polystyrene is provided only to increase the back pressure from a column comprising a CPG or other synthesis support.
  • oligonucleotides of different lengths may be constructed (e.g., a 20-mer constructed in one synthesis column may be completed and sit idle, while a 32-mer is constructed in a second synthesis column). Achieving successful purges after each liquid addition prevents liquid leakage (e.g. additional liquid reagent applied to a synthesis column that was not successfully purged will cause the column to overflow).
  • Figure 45 illustrates a computer system 62 coupled to the synthesizer 11.
  • the computer system 62 preferably provides the synthesizer 1, and specifically the controller 24, with operating instructions. These operating instructions may include, for example, rotating the cartridge 3 to a predetermined position, dispensing one of a plurality of reagents into selected synthesis columns through the valves 15 and dispense lines 6, flushing the first bank of synthesis columns 4 and/or the second bank of synthesis columns 5, and coordinating a timing sequence of these synthesizer functions.
  • U.S. Patent 5,865,224 to Ally et al. further demonstrates computer control of synthesis machines.
  • the computer system 62 allows a user to input data representing oligonucleotide sequences to form a polymer chain via a graphical user interface.
  • the computer system 62 instructs the synthesizer 1 to perform appropriate functions without any further input from the user.
  • the computer system 62 preferably includes a processor, an input device and a display.
  • the computer 62 can be configured as a laptop or a desktop, and may be operably connected to a network (e.g. LAN, internet, etc.).
  • the present invention provides alignment detectors for detecting the alignment of any of the components of the present invention, as desired.
  • an alarm or other signal is provided so that a user can assure proper alignment prior to further operation.
  • a processor operates a motor to adjust that alignment.
  • Alignment detectors find particular use in the present invention for assuring the alignment of any components that are involved in an exchange of liquid materials. For example, alignment of dispense lines and synthesis columns and alignment of drains and waste tubes should be monitored. Likewise, the tilt angle of the cartridge or any other component that should be parallel to the work surface can be monitored with alignment detectors.
  • each synthesis column 12 fits within the receiving hole 11 within the cartridge 3 and is intended to provide a pressure-tight seal around each synthesis column 12 within the cartridge 3.
  • Figure 46 illustrates three cross-sectional detailed views of the assembly 66 (the assembly comprising the cartridge 3, the drain plate gasket 43 and the drain plate 19) with a synthesis column 12 within a receiving hole 11 of cartridge 3. Each view shows a different embodiment of an airtight seal between the assembly 66 and the exterior surface 61 of synthesis column 12.
  • the airtight seal is provided by an O-ring 67.
  • the O-ring 67 is accessible for easy insertion and removal, e.g., for cleaning or replacement.
  • an O-ring 67 is positioned at the top of receiving hole 11, held in place by, e.g., a restraining plate 68, or any other suitable resfraining fitting.
  • a channel 69 is provided at the top of receiving hole 11 in cartridge 3 to accommodate the O-ring 67, as illustrated in Figure 46A.
  • a groove 70 within receiving hole 11 in cartridge 3 accommodates an O-ring 67, providing a groove lip 71 to restrain the O-ring 67, as illustrated in Figure 46B.
  • the groove lip 71 is about 0.030 inches.
  • Figure 46C illustrates a further embodiment, in which drain plate gasket 43 is configured to provide an airtight seal between nucleic acid synthesis column 12 and assembly 66.
  • the illustrations in Figure 46 are provided by way of examples only, and it is not intended that the present invention be limited by details of these illustrations, such as apparent size, shape or precise locations of features such as grooves, channels, plates or seals. Any O-ring configuration that helps maintain proper pressure differential across the synthesis columns is contemplated.
  • O-rings 67 may be composed of any suitable material, preferably a chemically resistant, resilient material that flexes upon engagement of the synthesis column 12 in receiving hole 11. fri some embodiments, a low cost material such as silicone or VITON may be used. In other embodiments, more expensive materials offering longer term stability, such as KALREZ, may be used. In some embodiments the O-rings may have a light lubrication, e.g. with a silicone or fluorinated grease.
  • the present invention provides a means of collecting emissions from reagent reservoirs 72 (See e.g., Figure 47A and B) by providing a reagent dispensing station.
  • the reagent dispensing station is an integral part of the base 2 of the synthesizer, as illusfrated in Figures 47 A and 47B.
  • the reagent dispensing station provides an enclosure for collecting emitted gasses.
  • the enclosure is created by the provision of a panel 73 to enclose a portion of base 2 containing reagent reservoirs 72, as illustrated in Figure 47B.
  • the panel 73 is movable for easy access to reagent reservoirs. In some embodiments, it is removeably attached.
  • Removable attachment may be accomplished by any suitable means, such as through the use of VELCRO, screws, bolts, pins, magnets, temporary adhesives, and the like.
  • at least a portion of the panel 73 is slidably moveable.
  • at least a portion of panel 73 is transparent.
  • the enclosure of the reagent dispensing station comprises a viewing window that is not in a panel 73.
  • the enclosure comprises a ventilation tube.
  • panel 73 comprises a ventilation port 74, e.g., for attachment to a ventilation tube. Since reagent vapors are typically heavier than air, in prefened embodiments, the ventilation tube is attached at the bottom for the enclosure. In a particularly prefened embodiment, the ventilation port is positioned toward the rear of the instrument.
  • the enclosure further comprises an air inlet.
  • a clearance 75 between the panel 73 and the base 2 provides an air inlet.
  • the air inlet is positioned toward the front of the instrument.
  • the location of the ventilation port 74 and air inlet is not limited to the panel 73.
  • the reagent dispensing station comprises a stand for holding the reagent bottles and a ventilation tube, wherein the stand holds the reagent reservoirs and the ventilation tube removes emitted gases. Ventilation may be continuous or under the control of an operator. For example, in some embodiments, when the panel 73 is in a closed position, ventilation occurs continuously through the ventilation port 74 or at regular intervals.
  • an operator may manually activate ventilation prior to opening the panel 73.
  • ventilation occurs in an automated fashion immediately prior to the opening of panel 73.
  • activation of the "open" routine triggers ventilation prior to the physical opening of panel 73.
  • the contents of the reagent containers are monitored by a sensor and the ventilation is triggered when one or more of the reagent containers are depleted.
  • the panel 73 is also automatically open, indicating the need for additional reagents and/or allowing an automated reagent container delivery system to supply reagents to the system.
  • the present invention also provides systems for ventilation, particularly ventilation of reaction enclosures (e.g., a chamber bowl 18), that improve the safety of synthesizers.
  • the ventilation systems of the present invention may be applied to any type of synthesizer, and preferably, to open type synthesizers. These systems are particularly useful for improving the function and safety of certain commercially available synthesizers, such as the ABI 3900 Synthesizer.
  • the present invention provides systems for collecting emissions from synthesizers without the use of a separate fume hood.
  • the present invention comprises a synthesizer having an integrated ventilation system to contain and remove vapor emissions.
  • the integrated ventilation system of the present invention is described as applied to the components and features of open synthesizers like the Applied Biosystems 3900 instrument. However, this configuration is used only as an example, and the integrated ventilation systems are not intended to be limited to the 3900 instrument or to any particular synthesizer.
  • One aspect of the invention is to collect and remove vapors when the instrument is open, e.g., for access by the operator to the reaction chamber ( Figures, 48C, and 49A-C).
  • the integrated ventilation system comprises a ventilated workspace.
  • Embodiments of an integrated ventilation system comprising a ventilated workspace as applied to the 3900 instrument are shown in Figures 48A-C, 49A-C and 50 A-B. Another embodiment is diagrammed in Figures 51 A and B.
  • a ventilation opening is provided through an opening in the top.
  • some embodiments of synthesizers of the present invention comprise a top enclosure (e.g. 97 ) that forms a primarily enclosed space 104 over a top cover (e.g., 30, not shown in this figure).
  • the top enclosure has four sides (e.g., 98, two of which are shown in Figure 48 A), and a top panel (e.g., 99) that form a primarily enclosed space 104 above the top cover (e.g., 30) containing a plurality of valves (e.g., 10, not shown in this figure) and a plurality of dispense lines (e.g., 6, not shown in this figure).
  • the top panel e.g., 99
  • the top panel contains an outer window (e.g., 101).
  • the outer window contains a ventilation opening (e.g., 105).
  • the combination of a top enclosure (e.g., 97) and top cover (e.g., 30) is refened to collectively as the “lid enclosure” (e.g., 102).
  • the "lid enclosure” has six sides, with the top cover (e.g., 30) serving as the "bottom", the top panel serving as the surface opposite the top cover, and the four side walls being the top enclosure sides (e.g., 98).
  • the lid enclosure has a ventilation opening (e.g., 105) with a ventilation tube (e.g., 103) attached thereto (See, Figure 48B).
  • the ventilation tube is connected to a ventilation opening in an outer window 101.
  • the synthesizer base (e.g., 2) comprises a primarily enclosed space 104.
  • a base (e.g., 2) of a synthesizer comprises a ventilation opening (e.g., 105) with a ventilation tube (e.g., 103) attached thereto (See, e.g., Figures 51A and 51B).
  • the ventilation openings in the lid enclosure or the base may be in any suitable position.
  • the ventilation opening in the lid enclosure may be in the top panel (e.g. in the center, toward the back of the machine, or in one of the corners).
  • the ventilation opening may also be located in a top enclosure side.
  • the ventilation opening may be in the enclosure side at the back of the machine, or on one of the sides (e.g., configured such that the lid enclosure may still be moved upward and downward while attached to a ventilation tube).
  • a ventilation opening in a base may be, for example, on the front, the sides or on the back (e.g., configured such that the lid enclosure may still be moved upward and downward without interference by the ventilation tube).
  • the ventilation opening is positioned toward the rear (e.g. , on a side or in the back) to allow the ventilation tubing to be directed away from an instrument operator.
  • the ventilation opening is on the back of the base, e.g., as shown in Figures 51 A and 5 IB.
  • the ventilation is located in a position such that air traveling through the primarily enclosed space (e.g., 104) make greater or less contact with particular synthesizer components located inside the lid enclosure (e.g. valves, solenoids, dispense lines, etc.).
  • the lid enclosures of the present invention may also have a plurality of ventilation openings. This may be desirable in order to control or direct air flow through the primarily enclosed space (e.g., to minimize or to maximize air contact with particular synthesizer components inside the lid enclosure).
  • the lid enclosure is hinged so that is may be moved upward and downward (e.g., allowing access to the chamber bowl or other reaction chamber by a user).
  • the primarily enclosed space of the lid enclosure (e.g. 104, not shown in this figure) is open to the ambient environment through a ventilation slot (e.g. 100) in the top cover or the top enclosure (e.g. in top enclosure side wall towards the back of the machine).
  • a lid enclosure is present on a commercially available machine (e.g., ABI 3900), and the lid enclosure is modified as described herein (e.g. , a ventilation opening is made in the lid enclosure)
  • An opening near the hinge for wiring serves as a ventilation slot on the 3900.
  • the lid enclosure must be added to synthesizer.
  • a synthesizer that simply has a top cover (e.g., 30), may have a top enclosure (e.g., 97) added thereto.
  • the lid enclosure is fabricated as a separate component, then installed onto a synthesizer.
  • the components making up the lid enclosure may be formed from a single mold, or two molds, etc.
  • features of the present invention may be built into the lid enclosure, such as the ventilation opening, ventilation slot, and certain hood components (described below).
  • the lid enclosure e.g., as diagrammed in Figures 48A-C, the lid enclosure (e.g.,
  • the lid enclosure comprises windows constructed of transparent or translucent material, such as plexiglass.
  • the lid enclosures of the present invention comprise a top panel directly opposite a top cover, and side walls between these two components
  • the primarily enclosed space between the top panel and top cover is, in some embodiments, open to the ambient environment through a ventilation slot near the lid enclosure hinge (e.g. , 106).
  • the lid enclosure of the present invention comprises an inner window and an outer window (e.g. an outer window in the top panel, and an inner window in the top cover).
  • the outer window of the instrument allows visual inspection of operations and components within the lid and within the chamber bowl 18 of the base 2.
  • the inner window seals the chamber bowl 18 by pressing against the chamber gasket when the lid enclosure is closed. Reagent supply tubing passes through the inner window, but the window is sealed around each tube so that the chamber will maintain appropriate pressure during operation.
  • the ventilation opening provides an aperture is the outer window.
  • the ventilation opening (e.g., 105) is attached to a ventilation tube (e.g., 103), that in turn may be attached to an exhaust system.
  • a synthesizer is attached to an individual exhaust system.
  • multiple synthesizers are attached to a centralized exhaust system (e.g. centralized venting or vacuum system).
  • access to the exhaust system is toward the rear of the instrument, to minimize or prevent interference by the ventilation tubing with operator access to the chamber bowl, and to conduct the fumes away from instrument operators.
  • the centralized exhaust may be a constant vacuum or a periodically actuated vacuum.
  • raising the top cover or hd enclosure of a synthesizer triggers the vacuum system.
  • reagent bottles on the sides of a synthesizer may also be vented through ventilation ports employing the same ventilation system employed by the ventilation tube attached to the top panel.
  • FIG. 49A One embodiment of a ventilated workspace is shown in Figure 49A, wherein the ventilated workspace is created by providing side panels (e.g., 107). Two variations of another embodiment are shown in Figures 49B and 49C.
  • the ventilated workspace is created by providing side panels (e.g., 107) between the body of the synthesizer and the lid enclosure, and a front panel (e.g., 108).
  • the ventilated workspace is created by including only side panels.
  • the ventilated workspace is created by only including a front panel.
  • side and front panels are used together (e.g., as in Figures 49B and 49C) to create a ventilated workspace.
  • side and front panels are provided as separate components.
  • a single component comprising both side panels and a front panel is provided.
  • the size of the ventilated workspace can be altered by the placement of the panels, e.g., the side panels (107) shown in Figures 49 A-C.
  • panels are positioned to maximize the size of the enclosed ventilated workspace (e.g., as in Figure 49B).
  • the panels are positioned to provide a smaller ventilated workspace (e.g., as with the side panels in Figure 49C).
  • the side panels are positioned as close to the top chamber gasket (e.g., 31) as they can be without disturbing the seal between the top chamber gasket and the top cover 30.
  • the front and or side panels are used with a synthesizer only having a top cover (not a full lid enclosure).
  • the side panels can be made of a number of different materials.
  • the materials used for the side panels are opaque.
  • the side panels are translucent or clear (e.g., to permit sunounding light into the ventilated workspace).
  • the side panels are constructed from flexible polymeric material (e.g., sheeting), such as polyethylene or polypropylene.
  • the polymeric material has an average thickness of about 2 to 8 mils. In prefened embodiments, the polymeric material has an average thickness of about 2 to 4 mils.
  • the panels are collapsible (i.e., can collapse or fold down upon themselves as the lid enclosure or top cover, is lowered).
  • panels are accordion-style or fan-fold style barriers that fold down upon themselves when the top cover or lid enclosure is lowered.
  • they when the panels are collapsed, they have a total thickness that is less than the height of the O-ring or gasket (e.g., top chamber seal 31) on the interior of the synthesizer (e.g., so that there is no interference with the sealing of the O-ring).
  • the side panels are constructed of rigid material.
  • rigid side panels are configured to fit into recesses in the body of the synthesizer when the top cover or lid enclosure is closed.
  • rigid side panels are configured to fit around the outside of the base of the synthesizer when the top cover or lid enclosure is closed.
  • rigid side panels are constructed from opaque materials (e.g., steel, aluminum, opaque plastic).
  • rigid side panels are constructed from translucent or fransparent material, such as plexiglass. Generally, the side panels are connected to the top cover, so when the top cover or lid enclosure is raised, the side panels slide up to form sides for the ventilated workspace.
  • a front panel (e.g., 108) is attached to the lid enclosure.
  • the front panel may attach to the top cover (e.g., Figure 49B), or the front panel may attach to one of sides of the lid enclosure (e.g., Figure 49C).
  • the front panel may drape over the front of the synthesizer when the lid enclosure is closed (See, e.g., Figures 48B and 49C).
  • the front panel may fit into a recessed slot in the synthesizer base, or fold up upon itself as the lid enclosure is lowered into the closed position.
  • Attachment of the panels provided for the pu ⁇ ose of enclosing the ventilated workspace is not limited to any particular means.
  • panels are attached by use of strips of VELCRO fastener (e.g., adhesive backed strips), for easy mounting and removal.
  • the panels may be attached using fasteners, including but not limited to screws, bolts, welds, and snaps, or may be attached with removable or permanent adhesives.
  • fasteners including but not limited to screws, bolts, welds, and snaps, or may be attached with removable or permanent adhesives.
  • the presence of the panels reduces the size of the opening through which ambient air can enter the ventilated workspace, and also reduces the size of the opening from which air and vapors in the chamber bowl can escape.
  • the ventilation system When the ventilation system is turned on (e.g., when the connected ventilation tube is drawing air from the ventilation opening, the airflow through the reduced opening prevents or reduces any flow (e.g. outward flow) of gaseous emissions.
  • the ventilation system When the ventilation system is actuated, ambient air and reagent vapors are drawn across the chamber bowl (e.g., 18) and into the ventilation slot (e.g., 100), as diagrammed in Figures 50B and 51B. The air and vapors then move through the primarily enclosed space (e.g., 104) and exit through the ventilation opening (e.g., 105) into the ventilation tube (e.g., 103).
  • the air flow rate at the opening of the ventilated workspace (e.g., in the embodiments shown in Figures 49B and 49C, where the sunounding air is drawn into the ventilated workspace below the front panel and between the side panels) is from about 20 to about 100 feet per minute, face velocity. In some prefened embodiments, the flow rate at the opening is about 40 to 50 feet per minute, face velocity.
  • the air and vapors may be vented, treated or collected.
  • the vented air and vapors are routed to a central scrubber.
  • the central scrubber may form part of an overall emission control system.
  • the central system may also be used to adjust total airflow for the number of synthesizers that are open at the same time. In this regard, exhaust from the system is minimized so as to concentrate waste vapors.
  • the size of the ventilation slot may be adjusted (e.g. reducing the size of the ventilation slot increase the speed of the moving air and vapors).
  • the airflow pattern made possible by the present invention allows synthesizers to be opened (e.g. to change columns, etc) without exposure of an operator, to hazardous vapors (e.g. argon, solvent fumes, etc).
  • the integrated chamber ventilation system of the present invention may be adapted to many synthesizers of both 'open' and 'closed' design.
  • another synthesizer that can be modified to include the reaction enclosure ventilation system of the present invention is the POLYPLEX 96-channel, high-throughput oligonucleotide synthesizer from GeneMachines, San Carlos, CA, which comprises a synthesis case providing an enclosure for the synthesis block in which the reactions are performed.
  • a similar instrument is described in WO 00/56445, published September 28, 2000, and in related U.S. Provisional Patent application 60/125262, filed March 19, 1999, each inco ⁇ orated herein in their entireties.
  • the synthesis case has a loading station, drain station, and water-tolerant and water- sensitive reagent filling stations.
  • the synthesis case has a cover, a first and a second side, a first and a second end, and a bottom side, which contacts the base.
  • the load station comprises a sealable opening in the synthesis case through wliich a multiwell plate can be inserted.
  • the synthesis case can be fitted with one or more ventilation openings similar to ventilation opening 105, for attachment to ventilation tubing (e.g., 103).
  • a ventilation opening is in a side of the synthesis case opposite the side having the sealable opening.
  • a ventilation opening in the synthesis case is on the first or second end.
  • the ventilation system is actuated when the sealable opening is opened, e.g., for insertion or removal of a multiwell plate.
  • the present invention also contemplates robotic means (e.g. conveyor belt, robots, etc) for linking the synthesizers to other components of the production process.
  • robotic means e.g. conveyor belt, robots, etc
  • Figure 52 illusfrates a synthesizer 1, a robotic means 92, a cleave and deprotect component 93 and a purification component 94 operably linked together.
  • the present invention provides synthesizer anays (e.g., groups of synthesizers).
  • the synthesizers are ananged in banks.
  • a given bank of synthesizers may be used to produce one set of oligonucleotides.
  • the present invention is not limited to any one synthesizer. Indeed, a variety of synthesizers are contemplated, including, but not limited to the synthesizers of the present invention, MOSS EXPEDITE 16-channel DNA synthesizers (PE Biosystems, Foster City, CA), OligoPilot (Amersham Pharmacia,), and the 3900 and 3948 48- Channel DNA synthesizers (PE Biosystems, Foster City, CA).
  • synthesizers are modified or are wholly fabricated to meet physical or performance specifications particularly prefened for use in the synthesis component of the present invention.
  • two or more different DNA synthesizers are combined in one bank in order to optimize the quantities of different oligonucleotides needed. This allows for the rapid synthesis (e.g., in less than 4 hours) of an entire set of oligonucleotides (all the oligonucleotide components needed for a particular assay, e.g., for detection of one SNP using an INVADER assay [Third Wave Technologies, Madison, WI]).
  • the DNA synthesizer component includes at least 100 synthesizers. In other embodiments, the DNA synthesizer component includes at least 200 synthesizers, fri still other embodiments, the DNA synthesizer component includes at least 250 synthesizers. In some embodiments, the DNA synthesizers are run 24 hours a day.
  • the NEI-48 synthesizer includes external mounting points for various reagent bottles, such as the phosphoramidite monomers used to form the polymer chain, and the oxidizers, capping reagents and deblocking reagents used in the reaction steps.
  • TEFLON tubing feeds liquid from each reagent bottle to its assigned valve on the top of the machine. The feeding is done under pressure from an argon gas source.
  • the operations of the machine are controlled using a computer.
  • the computer is fitted with a motion control card connected via cabling to a motor controller in the synthesizer; in addition, the computer is connected to the synthesizer via an RS-232C cable.
  • the provided software allows the user to monitor and control the machine's synthesis operations.
  • the machine also requires connection to a source of argon gas, to be delivered at a pressure between 15 and 60 psi, inclusive, and a source of compressed air or nitrogen, to be delivered at a pressure between 60 and 120 psi, inclusive.
  • a source of argon gas to be delivered at a pressure between 15 and 60 psi, inclusive
  • a source of compressed air or nitrogen to be delivered at a pressure between 60 and 120 psi, inclusive.
  • Operations of the NEI-48 in accordance with the manufacturer's instructions produced undesirable emissions and leakage resulting in potential synthesis and instrument failure.
  • the following section details two of the sources of these emissions, and details one or more aspects of the present invention applied to solve each problem, to thereby improve the performance of this machine.
  • the flow of reagent and waste from the synthesis columns is controlled by a differential in the pressure of argon between the top and bottom openings of the column.
  • the pressure of argon on the top opening is not sufficiently high, the column will not drain or be purged completely, i.e., fluid that should be drained will remain in the column.
  • This improper purging not only reduces the efficiency of the synthesis chemistry, it also leads to column overflow. Therefore, failure of either initial pressurization of the chamber, or leakage of argon from any coupling (in an amount great enough to reduce either the overall pressure of the system or the pressure differential across the synthesis column) may lead to undesirable emissions and exposure.
  • One aspect of the present invention is to prevent column overflow by reducing leakage of argon at a variety of points in the system.
  • the NEI-48 demonstrated a variety of failures as a result of argon leakage from or within the instrument.
  • the drain plate gasket 43 of the present invention was created and was fitted between the cartridge and drain plate. Addition of the gasket to this assembly, as diagramed in Figure 38, provided a pressure-tight seal, thereby containing the argon and allowing proper drainage of the columns at the purging step.
  • the gasket of the present invention applied in this way improved the safety of the machine, and improved the efficiency of the synthesis reaction.
  • a modified drain plate gasket was provided.
  • the drain plate has securing holes 33, for attachment of the motor connector 22.
  • the first gasket was of a design that avoided the areas of the motor connector 22 and the securing holes 33.
  • a modified drain plate gasket was designed with guide holes 44 to fit closely around each securing hole 33, such that the holes served to place the gasket in a specific position between the cartridge and the drain plate ( Figure 38).
  • the drain plate 19 and the cartridge 3 may be provided with other alignment features, such as pin fittings and conesponding pin receiving holes (not shown) to facilitate alignment of these parts during assembly (e.g., after cleaning).
  • a modified drain plate gasket for use with these parts may be provided with pin guide holes (not shown). Use of either the securing holes 33, or pins fittings to align the gasket makes the gasket easier to position during assembly, ensuring proper operation of the gasket and improving ease of any maintenance that requires disassembly of these parts.
  • reagent bottles During normal operations and without any malfunction, fumes can nonetheless be emitted by the reagent bottles attached to the machine. These emissions can be increased by poor fit or inconect seals around bottle caps.
  • the reagent bottles for the NEI-48 are affixed to the machine by clamps that apply pressure to the outside of the bottle caps. The clamps can distort the caps, increasing leakage and gaseous emissions.
  • One aspect of the present invention is to provide a means of collecting emissions from reagent bottles.
  • a reagent stand comprising a ventilation tube was constructed.
  • the stand holds the reagent bottles, thereby eliminating the need for the cap- distorting clamps, and consequently reducing emissions from the bottles; the ventilation tube removes any remaining emitted gases.
  • This reagent dispensing station improves the safety of the machine in normal operation.
  • the reagent dispensing station of the present invention is not limited to a configuration comprising a stand. It is envisioned that a station comprising a ventilation system may also be used with one or more bottles held in clamps, fri prefened embodiments, at least one aspect of the reagent container system, e.g., the clamp, the cap, or the bottle, is modified such that clamping the reagent bottle does not compromise the containment function of the cap, or of any other aspect of the reagent container system.
  • the Applied Biosystems 3900 Oligonucleotide Synthesizer (Applied Biosystems, Foster City, CA) is similar in design and function to the NEI-48, described above.
  • the 3900 is an "open system" synthesizer utilizing a round cartridge containing the columns.
  • the receiving holes of the cartridge are essentially cylindrical, and, as with the NEI-48, proper function of the instrument relies on an airtight seal between the columns and cartridge.
  • the 3900 synthesizer includes recessed areas for the external mounting of reagent bottles.
  • the reagent bottles When mounted on the instrument, the reagent bottles do not protrude beyond the outside edges of the instrument; they are completely recessed, (as, e.g., the reagent reservoirs 72 are recessed in base 2, diagrammed in Figure 47 A).
  • the reagent feeding is done under pressure from an argon gas source.
  • the performance of the 3900 synthesizer is improved using the modifications provided by the present invention. Two specific improvements are described below. These particular improvements are described by way of example; improvements to the ABI 3900 synthesizer, or any synthesizer, are not limited to the improvements described herein below.
  • the present invention provides improved synthesizers having reliable seals between the cartridge and the synthesis columns.
  • the present invention provides a number of embodiments of synthesizers having such seals.
  • a synthesizer may be improved by the addition of a resilient seal, such as an O- ring, in the receiving hole of each cartridge.
  • the 3900 is fitted with such O-rings for safer, more reliable and more efficient performance.
  • Examples of several means of creating an improved seal between the outer surface of a column 61 and a receiving hole 11 are diagrammed in Figures 46A-46C, While any of the embodiments of seals disclosed herein may be applied to the 3900 instrument, in a prefened embodiment, the 3900 is improved by the use of an embodiment similar to that diagrammed in Figure 46B, wherein a groove 70 creates a groove lip 71, to accommodate and hold an O-ring 67, thus providing a seal between cartridge 3 and the exterior surface 61 of the synthesis column 12.
  • the receiving hole 11 is enlarged in diameter to facilitate insertion and removal of an O-ring 67, e.g., for easy cleaning or replacement.
  • a groove is machined into the interior of each receiving hole in a 3900 cartridge, and appropriate O-ring seals are placed in the grooves.
  • the O-ring could be of any suitable material.
  • the cartridge of the 3900 has a greatly improved ability to accommodate imperfections in the exteriors of synthesis columns, and this improvement results in safer, and more efficient and reliable operation of the instrument, with fewer costs associated with chemical spill clean-up, instrument down-time, and the disposal of unusable synthesis columns.
  • the present invention provides a means of collecting emissions from reagent bottles.
  • the reagent bottles are attached in recessed areas on the exterior in the base of the instrument (e.g., the reagent reservoirs 72 attached to the recessed areas in the base 2, as illustrated in Figure 47 A).
  • the emissions from this instrument are reduced by modification to provide the enclosed reagent dispensing station of the present invention.
  • the recessed areas are provided with panels to enclose the space, reducing the release of hazardous vapors.
  • Reagent bottles or reservoirs need to be accessible for changing or filling, due, e.g., to consumption of reagents during synthesis operations.
  • the panels added to the instrument are moveable, to provide access to the reagent bottles within the enclosed space.
  • panels provided for the pu ⁇ ose of enclosing the space are attached by use of strips of VELCRO fastener (e.g., adhesive backed strips), for easy mounting and removal.
  • the panels may be attached using hard, removable fasteners, such as screws or bolts.
  • the panels are mounted in tracks, brackets or other suitable fittings that allow them to be moved or removed by sliding.
  • the panels are constructed such that the reagent bottles can be visually inspected without opening the enclosure.
  • the panels provided are constructed of transparent material. While glass may be used, in prefened embodiments, for both safety and ease of handling a plastic is used with sufficient transparency to allow visual inspection of reagent bottles, and with sufficient resistance to the chemicals used in synthesis to avoid rapid or immediate decay or fogging, (as is often associated with exposure of plastics to vapors of solvents to which they are not resistant), when used in this application. Selection of plastics for appropriate chemical resistance is well known in the art, and tables of chemical compatibility are generally readily available from manufacturers.
  • the panels are provided with a ventilation port (e.g., ventilation port 74, as diagrammed in Figure 47B), for the removal vapors and fumes emitted by the reagent bottles.
  • a ventilation port serves as an attachment point for a ventilation tube to conduct fumes away from the instrument, e.g., into an exhaust system. Since the vapors from DNA synthesis reagents tend to be heavier than air, the ventilation port is placed near the bottom of the enclosure. Placement of the ventilation port toward the rear is convenient for attachment to a larger exhaust system, minimizes or prevents interference by the ventilation tubing with operator access to other parts of the instrument, and conducts the fumes away from instrument operators.
  • an air inlet into the enclosure is provided.
  • a clearance between the attached panels and the body of the instrument e.g., the clearance 75 between the panel 73 and the base 2 diagrammed in Figure 47B
  • the panel is positioned such that the principal air inlet is a clearance between the front edge of the panel (i.e., the edge closest to the front of the instrument) and the instrument base.
  • the inward flow of air minimizes the possible escape of hazardous vapors toward instrument operators.
  • the 3900 instrument is improved with respect to its emissions of hazardous vapors.
  • the present invention provides a means of collecting emissions from the 3900 without the use of a separate fume hood.
  • the present invention comprises a synthesizer having an integrated ventilation system to contain and remove vapor emissions.
  • One aspect of the invention is to collect and remove vapors when the instrument is open.
  • Embodiments of integrated ventilation systems as applied to the 3900 instrument are shown in Figures 48-51.
  • the lid enclosure 102 is modified to comprise a ventilation opening 105.
  • the lid enclosure of the 3900 comprises an outer window 101.
  • a ventilation opening is placed in the center of the outer window 101 of the lid enclosure 105, so as to avoid blocking the operator's view of internal components, such as the synthesis columns, during operation.
  • the lid enclosure of the 3900 instrument comprises an outer window 101 and an inner window 25.
  • the space between the windows is open to the ambient environment through a ventilation slot 100 near the lid enclosure hinge 106.
  • the outer window in an unmodified instrument allows visual inspection of operations and components within the lid enclosure and within the chamber bowl 18 of the base 2.
  • Reagent supply tubing passes through the inner window, but the window is sealed around each tube so that the chamber will maintain appropriate pressure during operation.
  • the ventilation opening provides an aperture in the outer window.
  • one or more ventilation openings may be provided in the base (e.g., 2) of the synthesizer, as diagrammed in Figure 51.
  • a synthesizer may comprise ventilation openings in both a lid enclosure and a base.
  • Each ventilation opening is attached to ventilation tubing (e.g., 103) for attachment to an exhaust system.
  • a synthesizer is attached to an individual exhaust system.
  • multiple synthesizers are attached to a centralized exhaust system. In a prefened configuration, the access to the exhaust system is toward the rear of the instrument, to minimize or prevent interference by the ventilation tubing with operator access to the chamber bowl, and to conduct the fumes away from instrument operators.
  • Another aspect of the present invention is to provide a ventilated workspace around the
  • the ventilated workspace is designed to allow the instrument operator to reach into the space (e.g., to remove the synthesis columns) without turning off the ventilation system.
  • Embodiments of a ventilated workspace are shown in Figure 49 A-C.
  • the ventilated workspace is created by providing side panels between the body of the synthesizer and the lid enclosure, and a front panel. The presence of the panels reduces the size of the opening through which ambient air can enter the ventilated workspace.
  • the ventilation system is turned on (i.e., when the connected ventilation tube is drawing air from the ventilation opening, the airflow in through the reduced opening prevents or reduces any outward flow of gaseous emissions.
  • the present invention provides closed-system solid phase synthesizers that are suitable for use in large-scale polymer production facilities. Each synthesizer is itself capable of producing large volumes of polymers. Furthermore, the present invention provides systems for integrating multiple synthesizers into a production facility, to further increase production capabilities.
  • nucleic acid synthesizers have limited synthesis capacity.
  • the 3900 DNA Synthesizer (Applied Biosystem, Foster City, CA) is one of the most capable synthesizers and produces fewer than 10040-mer oligonucleotides in a typical day production run. Additional synthesizers are described in U.S. Pat. Nos. 5,744,102, 4,598,049, 5,202,418, 5,338,831, 5,342,585, 6,045,755, and 6,121,054, and PCT publication WO 01/41918, herein inco ⁇ orated by reference in their entireties.
  • the synthesizers of the present invention dramatically increase capacity, with some embodiments allowing over 2000 40-mer oligonucleotides to be produced per day (e.g., during a 16 hour production day) at a 1 ⁇ M scale.
  • These capacities are achieved through the use of multi- chamber reaction supports that allow parallel synthesis of polymers within each chamber.
  • three or more chambers e.g., comprising synthesis columns
  • 96 or more chambers are provided on a reaction support, permitting a plurality of different oligonucleotides to be simultaneously produced.
  • Each reaction chamber is associated with its own reagent dispenser such that reagents are delivered to each chamber substantially simultaneously rather than delivery reagents in sequence.
  • the synthesizer is a closed system during operation (i.e., reagent delivery to the chambers and waste removal from the chambers occurs in a continuous pathway that is isolated from the ambient environment).
  • An example of a closed system is illustrated in Figure 53.
  • the synthesizers have a minimum number of moving parts.
  • the reaction support is immobile.
  • the synthesizer provides additional polymer production capabilities.
  • the synthesizer is configured to conduct cleavage and deprotection of synthesized oligonucleotide.
  • the same reaction support is used for both synthesis and cleavage and deprotection.
  • the same reagent dispensers are used for both synthesis and cleavage and deprotection.
  • the reaction support does not move during both the synthesis and cleavage and deprotection processes (i.e., synthesis and cleavage and deprotection occur at the same location).
  • the synthesizer also provides an integrated purification component (e.g., using the same reaction support and/or reagent dispensers with or without movement of the reaction support). Any other production components described herein may also be integrated with the synthesizer. Prefened features of the synthesizers of the present invention include: single day
  • Reagent delivery to the synthesizer is achieved using a novel fluidics system.
  • all fluid transfers are desired to be closed system; that is, a closed fluid circuit exists from source to waste at any time reagents are being transfened.
  • the supply circuit remains coupled to the synthesis columns that are supported by the reaction support for all operations except, in some embodiments, during nucleic acid coupling reactions.
  • the circuit to a particular column or columns is disconnected to allow fluid transfer mechanisms to be used on other columns. While the fluid transfer is re-routed, the columns undergoing the coupling reaction need not be exposed to the ambient environment (i.e., a sealed delivery path may be maintained).
  • the target fluid transfer system is a pressurized supply with dispense confrol valves. Reagents flow to the reaction chambers upon opening of the control valves, driven by a pressure differential.
  • the reaction support contains waste channels configured to receive waste from the reaction chambers.
  • each channel is configured with its own waste channel (See e.g., Figure 53).
  • the waste channels preferably feed into a single waste disposal line.
  • the waste system is gravity driven.
  • a valve-controlled vacuum is used to eliminate waste.
  • waste lines are fitted with a trityl monitoring device, fri prefened embodiments, the waste line is fitted with a qualitative trityl monitoring device. For example, colorimetric analysis of effluent using a CCD camera or a similar device provides a yes/no answer on a particular detritylation level.
  • Valves used to control reagent delivery and/or waste removal may be under automated confrol.
  • a plurality of reagent dispensers are provided, wherein a reagent dispenser is provided for each reaction chamber.
  • the reagent dispensers provide each of the reagents necessary to support a synthesis reaction within the reaction chamber.
  • the reagent dispenser comprises a plurality of reagent delivery lines, each line providing a direct fluidic connection between the reagent dispenser and individual supply tanks for the different reagents (See e.g., Figure 53).
  • FIG. 54 An example of such a reagent dispenser (2) is shown in Figure 54 from both a side view ( Figure 54A) and a cross-sectional bottom view ( Figure 54B).
  • the side view shows a single reagent delivery line (3) penetrating a top surface (4) of the reagent dispenser (2).
  • a retention ring (5) is used to support the reagent delivery line (3).
  • the reagent delivery line (3) ends at a reagent reservoir (6) that is configured to receive reagents from each of the delivery lines.
  • a seal (7) forms a contact between the delivery line (3) and the reagent reservoir (6).
  • the center of the reagent reservoir (6) comprises a delivery aperture (8).
  • the delivery aperture (8) is in fluidic contact with a delivery channel (9), with a seal (10) forming a contact between the delivery channel (9) and the delivery aperture (8).
  • the delivery channel (9) passes through a bottom surface (11) of the reagent dispenser (2) and may positioned by a retention ring (12).
  • the cross-sectional bottom view shown in Figure 54B shows the presence of nine delivery lines (3) contained within the reagent dispenser (2). Each delivery line empties into the reagent reservoir (6), represented by the eight pronged star.
  • Figure 55A shows one prefened embodiment of the reagent dispenser (2), wherein the outer surface of the delivery channel (9) contains first (13) and second (14) ring seals configured to form an airtight or substantially airtight seal with one or more points on the interior surface of a synthesis column (15) or other reaction chamber (e.g., with reaction chambers present in a synthesizer or a cleavage and deprotection component; see, for example Figure 55B).
  • common reagent tanks supply reagents to all of the reaction chambers.
  • the reagents tanks may be contained within the synthesizer or may be external to the synthesizer. Where the tanks are provided with the synthesizer, they are preferably contained in a vented chamber to reduce the build-up of gaseous or liquid waste in and around the synthesizer.
  • common reagent tanks supply reagents to a plurality of synthesizers. Examples of such delivery systems are provided, below.
  • some of the reagents are supplied externally and some of the reagents are supplied at or in the synthesizer (e.g., amidites).
  • one or more of the reagents are processed, e.g., under vacuum, to remove dissolved gasses.
  • the synthesizer comprises a means of delivering energy to the reaction chambers to, for example, increase nucleic acid coupling reaction speed and efficiency, allowing increased production capacity.
  • the delivery of energy comprises delivering heat to the reaction chambers.
  • the use of heat allows the use of alternate synthesis chemistries and methods, e.g., the phosphate triester method, which has the advantages of using more stable monomer reagents for , synthesis, and of not using tefrazole or its derivatives as condensation catalysts.
  • Heat may be provided by a number of means, including, but not limited to, resistance heaters, visible or infrared light, microwaves, Peltier devices, transfer from fluids or gasses (e.g., via channels or a jacketed system).
  • heat generated by another component of a synthesis or production facility system e.g., during a waste neutralization step
  • heat is delivered through the use of one or more heated reagents. Delivery of heat to reaction chambers also comprises embodiments wherein heat is created within the reaction chamber, e.g. , by magnetic induction or microwave treatment. It is contemplated that heating may be accomplished through a combination of two or more different means.
  • the delivery of heat provides substantially uniform heating to two or more reaction chambers. In some embodiments, heating is carried out at a temperature in a range of about 20 °C to about 60 °C.
  • the present invention also provides methods for determining an optimum temperature for a particular coupling chemistry. For example, multiple synthesizers are run side-by-side with each machine run at a different temperature. Coupling efficiencies are measured and the optimum temperature for one or more incubations times are determined. In other embodiments, different amounts of heat are delivered to different reaction chambers within a single synthesizer, such that different reaction chemistries or protocols can be run at the same time.
  • the closed system of the present invention will be configured to tolerate variations in the system pressure (i.e., the pressure within the closed system) related to heating or other energy input to the system.
  • the system e.g., every component of the system and every junction or seal within the system
  • the system will be configured to withstand a range of pressures, e.g., pressures ranging from 0 to at least 1 atm, or about 15 psi. It is contemplated that pressures may be varied between different points within the system.
  • reagents and waste fluids are moved through the reaction chamber by use of a pressure differential between one end (e.g., an input aperture) and the other (e.g., a drain aperture) of the reaction chamber.
  • the system of the present invention is configured to use pressure differentials within a pressurized system (e.g., wherein a system segment having lower pressure than another system segment nonetheless has higher pressure than the environment outside the closed system).
  • the prevention of backward flow of reagents through the system is controlled by use of pressure.
  • valves are provided to assist in control of the direction of flow,
  • the synthesizer comprises a mixing component configured to mix reaction components, e.g., to facilitate the penetration of reagents into the pores of the solid support.
  • Mixing may be accomplished by a number of means. In some embodiments, mixing is accomplished by forced movement of the fluid through the matrix (e.g., moving it back and forth or circulating it through the matrix using pressure and/or vacuum, or with a fluid oscillator). Mixing may also be accomplished by agitating the contents of the reaction chamber (e.g., stirring, shaking, continuous or pulsed ultra or subsonic waves, See, Figures 42A-C and 43 A and B). In some prefened embodiments, an agitator is used that avoids the creation of standing waves in the reaction mixture.
  • the agitator is configured to utilize a reaction vessel surface or reaction support surface (e.g., a surface of a synthesis column) to serve as resonant members to transfer energy into fluid within a reaction mixture.
  • the matrix is an active component of the mixing system.
  • the matrix comprises paramagnetic particles that may be moved through the use of magnets to facilitate mixing.
  • the matrix is an active component of both mixing and heating systems (e.g., paramagnetic particles may be agitated by magnetic control and heated by magnetic induction). It is contemplated that any of these mixing means may be used as the sole means of mixing, or that these mixing components may be used in combination, either simultaneously or in sequence.
  • the heating component and the mixing component are under automated control.
  • a central confrol processor is used to automate one or more of the synthesis steps or synthesizer operations.
  • the central control processor may also be configured to interact with one or more other components of a production facility (See below).
  • the central control processor regulates valves, controlling the timing, volume, a rate of reagent delivery to the reaction chambers, fri prefened embodiments, all delivered reagents are controllable for volume within prescribed ranges at each step of the synthesis process within a protocol independent of other steps.
  • the present invention is not limited by the range of flow rate used for reagent delivery. However, in prefened embodiments, flow rates are 300-500 ⁇ L/sec for all reagents.
  • Table 1 provides an example of reagent delivery times (in seconds) and amounts (in microliters) for a single synthesis cycle. Conditions are provided for four different synthesis scales.
  • reaction or wash times are controlled by fluid application rate without additional dwell time prior to purging. This is in contrast to methods used with cunent commercial synthesizers (e.g., 3900 DNA Synthesizers).
  • Batch size is preferably 96 anayed reaction chambers in a standard microtiter footprint.
  • Synthesis columns could be either independently filled and inserted into a rack to form the anay or, preferably, molded in an anayed format and filled as a batch. If the latter, then all columns should be of a similar type and synthesis operations are grouped accordingly.
  • Column plates are loaded one at a time and replaced at the end of the synthesis process.
  • loading and unloading is manual-no transport mechanisms required. In other embodiments, loading and unloading is controlled robotically. Fluid connections from the system to the column fray is either established by the system (moving mechanism) or by the user en mass (fixed dispense).
  • reagents are accomplished by a fixed set of multifunctional reagent dispensers, each inco ⁇ orating all required reagents: each column has a dedicated multiplexed supply line and no motion devices or fluid connection make/break cycles are required.
  • This approach requires a large number of valves (approximately 1000) and is therefore preferably uses very compact, relatively inexpensive and relatively high reliability valves.
  • This system is similar in configuration to the non-dedicated fluidics batch system described above, but allows multiple plate positions with the system. Walkaway time improves linearly with the number of plates allowed, throughput and other comments are similar. At increasing levels of resident plates, parallel (400 valve system) with 4 plates resident for each parallel line would allow walk away time of 5 hours. In principle, 4 runs of 8 plates could be completed per day producing 3072 oligonucleotides. A 200-valve system configured similarly could produce 1536.
  • This system is similar to the above system with the addition of queues for feeding plates and accumulating completed plates.
  • the system requires similar fluid handling but adds plate transport mechanisms.
  • the waste system is more complicated due to plate movement.
  • This system allows direct integration to downstream cleave and deprotect system and allows direct integration to synthesis column packing upstream. Throughput is slightly higher than the modified batch system.
  • the columns are prepared and presented in strips of 12 columns.
  • the strips are fed through multiple parallel reagent delivery ports. This approach allows greater spacing between adjacent fluidic elements and allows processing of multiple different column types simultaneously. An additional benefit is the likelihood that a closer approach to the theoretical maximum throughput should be routinely achieved. In this embodiment, throughput per valve would be similar to continuous batch, but tubing of throughput is easier.
  • the synthesizers of the present invention also provide components to reduce or eliminate undesired emissions.
  • a problem with currently available synthesizers is the emission of undesirable gaseous or liquid materials that pose health, environmental, and explosive hazards. Such emissions result from both the normal operation of the instrument and from instrument failures. Emissions that result from instrument failures cause a reduction or loss of synthesis efficiency and can provoke further failures and/or complete synthesizer failure. Conection of failures may require taking the synthesizer off-line for cleaning and repair.
  • the present invention provides nucleic acid synthesizers with components that reduce or eliminate unwanted emissions and that compensate for and facilitate the removal of unwanted emissions, to the extent that they occur at all.
  • the present invention also provides waste handling systems to eliminate or reduce exposure of emissions to the users or the environment. Such systems find use with individual synthesizers, as well as in large-scale synthesis facilities comprising many synthesizers (e.g. anays of synthesizers).
  • oligonucleotide synthesis involves the use of an array of hazardous materials, including but not limited to methylene chloride, pyridine, acetic anhydride, 2,6-lutidine, acetonitrile, tefr-ihydrofurane, and toluene.
  • hazardous materials including but not limited to methylene chloride, pyridine, acetic anhydride, 2,6-lutidine, acetonitrile, tefr-ihydrofurane, and toluene.
  • These reagents can have a variety of harmful effects on those who may be exposed to them. They can be mildly or extremely irritating or toxic upon short-term exposure; several are more severely toxic and/or carcinogenic with long-term exposure. Many can create a fire or explosion hazard if not properly contained.
  • many of these chemicals must be assessed for emissions from normal operations, e.g for determining compliance with OSHA or environmental agency standards. Malfunction of a system, e.g.,
  • Emission or leakage of reagents during operation can have consequences beyond risks to personnel and to the environment.
  • instruments may need to be removed from operation for cleaning, leading to a temporary decrease in production capacity of a synthesis facility.
  • any emission or leakage may cause damage to parts of the instrument or to other instruments or aspects of the facility, necessitating repair or replacement of any such parts or aspects, increasing the time and cost of bringing an instrument back into operation.
  • the synthesizers of the present invention provide a number of novel features that dramatically improve synthesizer performance and safety compared to available synthesizers. These novel features work both independently and in conjunction to provide enhanced performance. For example, the present invention reduces exposure by improving collection and disposal of emissions that occur during the normal operation of various synthesis instruments. In another embodiment, the present invention reduces exposure by improving aspects of the instrument to reduce risk of malfunctions leading to reagent escape from the system, e.g., through leakage, overflow or other spillage.
  • the present invention provides a means of collecting emissions from the interior of synthesizers by providing a reagent dispensing station.
  • the reagent dispensing station is an integral part of the base 2 of the synthesizer, as illusfrated in Figures 47A and 47B.
  • the reagent dispensing station provides an enclosure for collecting emitted gasses.
  • the enclosure is created by the provision of a panel 73 to enclose a portion of base 2 containing reagent reservoirs 72, as illustrated in Figure 47B.
  • the panel 73 is movable for easy access to reagent reservoirs, fri some embodiments, it is removeably attached.
  • Removable attachment may be accomplished by any suitable means, such as through the use of VELCRO, screws, bolts, pins, magnets, temporary adhesives, and the like.
  • at least a portion of the panel 18 is slidably moveable.
  • at least a portion of panel 18 is transparent.
  • the enclosure of the reagent dispensing station comprises a viewing window that is not in a panel 73.
  • the enclosure comprises ventilation tubing.
  • panel 73 comprises a ventilation port 74, e.g., for attachment to ventilation tubing. Since reagent vapors are typically heavier than air, in prefened embodiments, the ventilation tubing is attached at the bottom for the enclosure. In a particularly prefened embodiment, the ventilation port is positioned toward the rear of the instrument.
  • the enclosure further comprises an air inlet.
  • a clearance 75 between the panel 73 and the base 2 provides an air inlet.
  • the air inlet is positioned toward the front of the instrument.
  • the location of the ventilation port 74 and air inlet is not limited to the panel 73.
  • the reagent dispensing station comprises a stand for holding the reagent bottles and ventilation tubing, wherein the stand holds the reagent reservoirs and the ventilation tubing removes emitted gases. Ventilation may be continuous or under the control of an operator. For example, in some embodiments, when the panel 73 is in a closed position, ventilation occurs continuously through the ventilation port 74 or at regular intervals.
  • an operator may manually activate ventilation prior to opening the panel 73.
  • ventilation occurs in an automated fashion immediately prior to the opening of panel 73.
  • activation of the "open" routine triggers ventilation prior to the physical opening of panel 73.
  • the contents of the reagent containers are monitored by a sensor and the ventilation is triggered when one or more of the reagent containers are depleted.
  • the panel 73 is also automatically open, indicating the need for additional reagents and/or allowing an automated reagent container delivery system to supply reagents to the system.
  • multiwell plates e.g. 96 well, 384 well, 1536 well, etc
  • the synthesizers are parts of a full automated process such that oligonucleotides are produced without human interaction.
  • the oligonucleotides move through the synthesis component, and processing components, on rails.
  • the DNA synthesizers in the oligonucleotide synthesis component further comprise an automated reagent supply system.
  • the automated reagent supply system delivers reagents necessary for synthesis to the synthesizers from a central supply area.
  • the central supply area is provided in an isolated room equipped for accommodating leakage, fires, and explosions without threatening other portions of the synthesis facility, the environment, or humans.
  • the system is configured to allow banks of synthesizer or individual synthesizer to be removed from the system (e.g. , for maintenance or repair) without interrupting activity at other synthesizers.
  • the present invention provides an efficient fail-safe reagent delivery system.
  • acetonitrile is supplied via tubing (e.g., stainless steel or TEFLON tubing) through the automated supply system.
  • De-blocking solution may also be supplied directly to DNA synthesizers through tubing.
  • the reagent supply system tubing is designed to connect directly to the DNA synthesizers without modifying the synthesizers.
  • the central reagent supply is designed to deliver reagents at a constant and controlled pressure. The amount of reagent circulating in the cenfral supply loop is maintained at 8 to 12 times the level needed for synthesis in order to allow standardized pressure at each instrument. The excess reagent also allows new reagent to be added to the system without shutting down.
  • the DNA synthesis component includes a centralized argon delivery system.
  • the system includes high-pressure argon tanks adjacent to each bank of synthesizers. These tanks are connected to large, main argon tanks for backup.
  • the main tanks are. run in series, fri other embodiments, the main tanks are set up in banks.
  • the system further includes an automated tank switching system.
  • the argon delivery system further comprises a tertiary backup system to provide argon in the case of failure of the primary and backup systems.
  • one or more branched delivery components are used between the reagent tanks and the individual synthesizers or banks of synthesizers.
  • acetonitrile is delivered through a branched metal structure (e.g., the structure described in Figure 56).
  • each branched delivery component is individually pressurized.
  • each branched delivery component (100) contains ten or more branches (101).
  • Reagent tanks may be connected to the branched delivery components using any number of configurations. For example, in some embodiments, a single reagent tank is matched with a single branched component. In other embodiments, a plurality of reagent tanks is used to supply reagents to one or more branched components.
  • the plurality of tanks may be attached to the branched components through a single feed line, wherein one or a subset of the tanks feeds the branched components until empty (or substantially empty), whereby a second tank or subset of tanks is accessed to maintain a continuous supply of reagent to the one or more branched components.
  • an ultrasonic level sensor may be applied to automate the monitoring and switching of tanks.
  • each branch of the branched delivery component provides reagent to one synthesizer or to a bank of synthesizers through connecting tubing (102).
  • tubing is continuous (i.e., provides a direct connection between the delivery branch and the synthesizer).
  • the tubing comprises an interior diameter of 0.25 inches or less (e.g., 0.125 inches).
  • each branch contains one or more valves (preferably one). While the valve may be located at any position along the delivery line, in prefened embodiments, the valve is located in close proximity to the synthesizer.
  • reagent is provided directly to synthesizers without any joints or valves between the branched delivery component and the synthesizers.

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Abstract

La présente invention concerne la mise au point, la production, et l'optimisation de dosages de détection. En particulier, cette invention concerne des systèmes et des procédés permettant de collecter et d'analyser des informations biologiques. La présente invention concerne également la production d'un dosage de détection à l'aide de systèmes de traitement et de synthèse des oligonucléotides améliorés. Cette invention concerne également des systèmes intégrant la collecte d'informations biologiques à la production de dosages de détection permettant la mise au point rapide de produits commerciaux, tels que des réactifs propres à un échantillon à analyser et des diagnostiques in vitro.
PCT/US2001/045705 2000-11-30 2001-11-30 Systemes et procedes de commande, de conception, de production, d'inventaire, de vente et d'analyse de dosages de detection, pouvant etre utilises avec ou dans un moyen de production WO2002044994A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU3945502A AU3945502A (en) 2000-11-30 2001-10-31 Systems and methods for detection assay ordering, design, production, inventory,sales and analysis for use with or in_aproduction facility
EP01987217A EP1364334A2 (fr) 2000-11-30 2001-11-30 Systemes et procedes de commande, de conception, de production, d'inventaire, de vente et d'analyse de dosages de detection, pouvant etre utilises avec ou dans un moyen de production
AU2002239455A AU2002239455A1 (en) 2000-11-30 2001-11-30 Systems and methods for detection assay ordering, design, production, inventory, sales and analysis for use with or in a production facility
JP2002547086A JP2004536562A (ja) 2000-11-30 2001-11-30 増幅プライマーおよびプールされた試料における突然変異の検出

Applications Claiming Priority (49)

Application Number Priority Date Filing Date Title
US25044900P 2000-11-30 2000-11-30
US25011200P 2000-11-30 2000-11-30
US60/250,449 2000-11-30
US60/250,112 2000-11-30
US09/771,332 2001-01-26
US09/771,332 US6932943B1 (en) 2001-01-26 2001-01-26 Nucleic acid synthesizers
US78270201A 2001-02-13 2001-02-13
US09/782,702 2001-02-13
US28589501P 2001-04-23 2001-04-23
US60/285,895 2001-04-23
US28822901P 2001-05-02 2001-05-02
US60/288,229 2001-05-02
US28976401P 2001-05-09 2001-05-09
US60/289,764 2001-05-09
US30452101P 2001-07-11 2001-07-11
US60/304,521 2001-07-11
US30766001P 2001-07-25 2001-07-25
US09/915,063 US20030082544A1 (en) 2001-07-11 2001-07-25 Methods and systems for validating detection assays, developing in-vitro diagnostic DNA or RNA analysis products, and increasing revenue and/or profit margins from in-vitro diagnostic DNA or RNA analysis assays
US60/307,660 2001-07-25
US09/915,063 2001-07-25
US30887801P 2001-07-31 2001-07-31
US60/308,878 2001-07-31
US31158201P 2001-08-10 2001-08-10
US60/311,582 2001-08-10
US09/929,135 2001-08-14
US09/929,135 US20030104470A1 (en) 2001-08-14 2001-08-14 Electronic medical record, library of electronic medical records having polymorphism data, and computer systems and methods for use thereof
US09/930,535 2001-08-15
US09/930,688 US20030124526A1 (en) 2001-08-15 2001-08-15 Polymer synthesizer
US09/930,543 US20030113236A1 (en) 2001-08-15 2001-08-15 Polymer synthesizer
US09/930,646 US20030113237A1 (en) 2001-08-15 2001-08-15 Polymer synthesizer
US09/930,543 2001-08-15
US09/930,535 US20030072689A1 (en) 2001-08-15 2001-08-15 Polymer synthesizer
US09/930,688 2001-08-15
US09/930,646 2001-08-15
US32654901P 2001-10-02 2001-10-02
US60/326,549 2001-10-02
US23831201P 2001-10-10 2001-10-10
US60/238,312 2001-10-10
US32911301P 2001-10-12 2001-10-12
US32886101P 2001-10-12 2001-10-12
US60/328,861 2001-10-12
US60/329,113 2001-10-12
US36048901P 2001-10-19 2001-10-19
US60/360,489 2001-10-19
US10/002,251 2001-10-26
US10/002,251 US20020156255A1 (en) 2001-01-26 2001-10-26 Nucleic acid synthesizers
US10/054,023 2001-11-13
US10/054,023 US7435390B2 (en) 2001-01-26 2001-11-13 Nucleic acid synthesizers
USNONE 2006-11-06

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WO2002044994A9 WO2002044994A9 (fr) 2010-09-23

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1468103A1 (fr) * 2002-01-25 2004-10-20 Applera Corporation Kits d'analyse a tube unique, pret a l'emploi, et procedes d'utilisation associes
WO2005056837A2 (fr) * 2003-11-26 2005-06-23 Applera Corporation Polymorphismes genetiques associes a des troubles cardiovasculaires et a une reponse au medicament, leurs procedes de detection et d'utilisation
EP1567540A2 (fr) * 2002-11-14 2005-08-31 Third Wave Technologies, Inc. Dosages de detection d'alleles de cftr
WO2006060200A1 (fr) * 2004-11-30 2006-06-08 Agilent Technologies, Inc. Systèmes et procédés pour produire des agencements d’ensembles chimiques
EP1799865A2 (fr) * 2004-09-30 2007-06-27 Vanda Pharmaceuticals Inc. Methodes d'administration d'iloperidone
US7818181B2 (en) 2005-10-31 2010-10-19 Focused Medical Analytics Llc Medical practice pattern tool
WO2014015186A1 (fr) * 2012-07-18 2014-01-23 Labminds Ltd. Distributeur de solution automatique
WO2015054234A1 (fr) * 2013-10-07 2015-04-16 The University Of Chicago Système et méthodes de prescription génomique
US10286371B2 (en) 2011-01-21 2019-05-14 Labminds Ltd Automated solution dispenser
CN111798178A (zh) * 2020-06-24 2020-10-20 霓检有限公司 快递寄送方法、装置和电商服务器
WO2021168467A3 (fr) * 2020-02-18 2021-09-23 Blue Goji Llc Système et méthode d'évaluation, de détection, de conditionnement et de traitement du fonctionnement et de troubles neurologiques

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2553672A (en) 2015-02-06 2018-03-14 Labminds Ltd Automated solution dispenser

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
No Search *

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9464320B2 (en) 2002-01-25 2016-10-11 Applied Biosystems, Llc Methods for placing, accepting, and filling orders for products and services
US10689692B2 (en) 2002-01-25 2020-06-23 Applied Biosystems, Llc Methods for placing, accepting, and filling orders for products and services
EP1706826A2 (fr) * 2002-01-25 2006-10-04 Applera Corporation Procedes permettant de placer, d'accepter et de classer des commandes relatives a des produits et des services
EP1468103A1 (fr) * 2002-01-25 2004-10-20 Applera Corporation Kits d'analyse a tube unique, pret a l'emploi, et procedes d'utilisation associes
EP1706826A4 (fr) * 2002-01-25 2008-01-30 Applera Corp Procedes permettant de placer, d'accepter et de classer des commandes relatives a des produits et des services
EP1468103A4 (fr) * 2002-01-25 2008-12-31 Applera Corp Kits d'analyse a tube unique, pret a l'emploi, et procedes d'utilisation associes
EP1567540A2 (fr) * 2002-11-14 2005-08-31 Third Wave Technologies, Inc. Dosages de detection d'alleles de cftr
EP1567540A4 (fr) * 2002-11-14 2008-02-27 Third Wave Tech Inc Dosages de detection d'alleles de cftr
WO2005056837A2 (fr) * 2003-11-26 2005-06-23 Applera Corporation Polymorphismes genetiques associes a des troubles cardiovasculaires et a une reponse au medicament, leurs procedes de detection et d'utilisation
WO2005056837A3 (fr) * 2003-11-26 2006-10-05 Applera Corp Polymorphismes genetiques associes a des troubles cardiovasculaires et a une reponse au medicament, leurs procedes de detection et d'utilisation
EP1799865A2 (fr) * 2004-09-30 2007-06-27 Vanda Pharmaceuticals Inc. Methodes d'administration d'iloperidone
US10272076B2 (en) 2004-09-30 2019-04-30 Vanda Pharmaceuticals, Inc. Methods for the administration of iloperidone
EP2479290A3 (fr) * 2004-09-30 2012-10-10 Vanda Pharmaceuticals Inc. Procédés pour l'administration de l'ilopéridone
US8586610B2 (en) 2004-09-30 2013-11-19 Vanda Pharmaceuticals, Inc. Methods for the administration of iloperidone
EP1799865B1 (fr) * 2004-09-30 2012-06-06 Vanda Pharmaceuticals Inc. Methodes d'administration d'iloperidone
US9138432B2 (en) 2004-09-30 2015-09-22 Vanda Pharmaceuticals, Inc. Methods for the administration of iloperidone
WO2006060200A1 (fr) * 2004-11-30 2006-06-08 Agilent Technologies, Inc. Systèmes et procédés pour produire des agencements d’ensembles chimiques
US7818181B2 (en) 2005-10-31 2010-10-19 Focused Medical Analytics Llc Medical practice pattern tool
US10286371B2 (en) 2011-01-21 2019-05-14 Labminds Ltd Automated solution dispenser
GB2519890A (en) * 2012-07-18 2015-05-06 Labminds Ltd Automated solution dispenser
GB2519890B (en) * 2012-07-18 2019-01-16 Labminds Ltd Automated solution dispenser
WO2014015186A1 (fr) * 2012-07-18 2014-01-23 Labminds Ltd. Distributeur de solution automatique
WO2015054234A1 (fr) * 2013-10-07 2015-04-16 The University Of Chicago Système et méthodes de prescription génomique
WO2021168467A3 (fr) * 2020-02-18 2021-09-23 Blue Goji Llc Système et méthode d'évaluation, de détection, de conditionnement et de traitement du fonctionnement et de troubles neurologiques
CN111798178A (zh) * 2020-06-24 2020-10-20 霓检有限公司 快递寄送方法、装置和电商服务器

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AU2002239455A8 (en) 2006-12-14
WO2002044994A3 (fr) 2006-11-02
WO2002044994A9 (fr) 2010-09-23
EP1364334A2 (fr) 2003-11-26
WO2002044994A8 (fr) 2003-09-18

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